Lyophilized therapeutic peptibody Formulations

ABSTRACT

The present invention provides long-term stable formulations of a lyophilized therapeutic peptibody and methods for making a lyophilized composition comprising a therapeutic peptibody.

FIELD OF THE INVENTION

Generally, the invention relates to formulations of lyophilizedtherapeutic peptibodies and methods for making a lyophilized compositioncomprising therapeutic peptibodies.

BACKGROUND OF THE INVENTION

Recombinant proteins are an emerging class of therapeutic agents. Suchrecombinant therapeutics have engendered advances in protein formulationand chemical modification. Modifications have been identified that canprotect therapeutic proteins, primarily by blocking their exposure toproteolytic enzymes. Protein modifications may also increase thetherapeutic protein's stability, circulation time, and biologicalactivity. A review article describing protein modification and fusionproteins is Francis (1992), Focus on Growth Factors 3:4-10 (Mediscript,London), which is hereby incorporated by reference.

One useful modification is combination of a polypeptide with an “Fc”domain of an antibody. Antibodies comprise two functionally independentparts, a variable domain known as “Fab,” which binds antigen, and aconstant domain known as “Fc,” which links to such effector functions ascomplement activation and attack by phagocytic cells. An Fc has a longserum half-life, whereas an Fab is short-lived. Capon et al. (1989),Nature 337: 525-31. See also, e.g., U.S. Pat. No. 5,428,130. Whenconstructed together with a therapeutic peptibody or protein, an Fcdomain can provide longer half-life or incorporate such functions as Fcreceptor binding, protein A binding, complement fixation and perhapseven placental transfer. Id. Table 1 summarizes use of Fc fusions withtherapeutic proteins known in the art.

TABLE 1 Fc fusion with therapeutic proteins Fusion Therapeutic Form ofFc partner implications Reference IgG1 N-terminus of Hodgkin's disease;U.S. Pat. No. 5,480,981 CD30-L anaplastic lymphoma; T-cell leukemiaMurine Fcγ2a IL-10 anti-inflammatory; Zheng et al. (1995), J. transplantrejection Immunol. 154: 5590-600 IgG1 TNF receptor septic shock Fisheret al. (1996), N. Engl. J. Med. 334: 1697-1702; Van Zee, K. et al.(1996), J. Immunol. 156: 2221-30 IgG, IgA, IgM, TNF receptorinflammation, autoimmune U.S. Pat. No. 5,808,029, or IgE disordersissued Sep. 15, 1998 (excluding the first domain) IgG1 CD4 receptor AIDSCapon et al. (1989), Nature 337: 525-31 IgG1, N-terminus anti-cancer,antiviral Harvill et al. (1995), IgG3 of IL-2 Immunotech. 1: 95-105 IgG1C-terminus of osteoarthritis; WO 97/23614, published OPG bone densityJul. 3, 1997 IgG1 N-terminus of anti-obesity PCT/US 97/23183, filedleptin Dec. 11, 1997 Human Ig Cγ1 CTLA-4 autoimmune disorders Linsley(1991), J. Exp. Med. 174: 561-9

Polyethylene glycol (“PEG”) conjugated or fusion proteins and peptideshave also been studied for use in pharmaceuticals, on artificialimplants, and other applications where biocompatibility is ofimportance. Various derivatives of PEG have been proposed that have anactive moiety for permitting PEG to be attached to pharmaceuticals andimplants and to molecules and surfaces generally. For example, PEGderivatives have been proposed for coupling PEG to surfaces to controlwetting, static buildup, and attachment of other types of molecules tothe surface, including proteins or protein residues.

In other studies, coupling of PEG (“PEGylation”) has been shown to bedesirable in overcoming obstacles encountered in clinical use ofbiologically active molecules. Published PCT Publication No. WO 92/16221states, for example, that many potentially therapeutic proteins havebeen found to have a short half life in blood serum.

PEGylation decreases the rate of clearance from the bloodstream byincreasing the apparent molecular weight of the molecule. Up to acertain size, the rate of glomerular filtration of proteins is inverselyproportional to the size of the protein. The ability of PEGylation todecrease clearance, therefore, is generally not a function of how manyPEG groups are attached to the protein, but the overall molecular weightof the conjugated protein. Decreased clearance can lead to increasedefficacy over the non-PEGylated material. See, for example, Conforti etal., Pharm. Research Commun. vol. 19, pg. 287 (1987) and Katre et.,Proc. Natl. Acad. Sci. U.S.A. vol. 84, pg. 1487 (1987).

In addition, PEGylation can decrease protein aggregation, (Suzuki etal., Biochem. Biophys. Acta vol. 788, pg. 248 (1984)), alter (i.e.)protein immunogenicity (Abuchowski et al., J. Biol. Chem. vol. 252 pg.3582 (1977)), and increase protein solubility as described, for example,in PCT Publication No. WO 92/16221.

In general, the interaction of a protein ligand with its receptor oftentakes place at a relatively large interface. However, as demonstrated inthe case of human growth hormone bound to its receptor, only a few keyresidues at the interface actually contribute to most of the bindingenergy. Clackson, T. et al., Science 267:383-386 (1995). Thisobservation and the fact that the bulk of the remaining protein ligandserves only to display the binding epitopes in the right topology makesit possible to find active ligands of much smaller size. Thus, moleculesof only “peptide” length as defined herein can bind to the receptorprotein of a given large protein ligand. Such peptides may mimic thebioactivity of the large protein ligand (“peptide agonists”) or, throughcompetitive binding, inhibit the bioactivity of the large protein ligand(“peptide antagonists”).

Phage display peptide libraries have emerged as a powerful method inidentifying such peptide agonists and antagonists. See, for example,Scott et al. (1990), Science 249: 386; Devlin et al. (1990), Science249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29, 1993; U.S. Pat. No.5,733,731, issued Mar. 31, 1998; U.S. Pat. No. 5,498,530, issued Mar.12, 1996; U.S. Pat. No. 5,432,018, issued Jul. 11, 1995; U.S. Pat. No.5,338,665, issued Aug. 16, 1994; U.S. Pat. No. 5,922,545, issued Jul.13, 1999; WO 96/40987, published Dec. 19, 1996; and WO 98/15833,published Apr. 16, 1998, each of which is incorporated by reference. Insuch libraries, random peptide sequences are displayed by fusion withcoat proteins of filamentous phage. Typically, the displayed peptidesare affinity-eluted against an antibody-immobilized extracellular domainof a receptor. The retained phages may be enriched by successive roundsof affinity purification and repropagation, and the best bindingpeptides are sequenced to identify key residues within one or morestructurally related families of peptides. See, e.g., Cwirla et al.(1997), Science 276: 1696-9, in which two distinct families wereidentified. The peptide sequences may also suggest which residues may besafely replaced by alanine scanning or by mutagenesis at the DNA level.Mutagenesis libraries may be created and screened to further optimizethe sequence of the best binders. Lowman (1997), Ann. Rev. Biophys.Biomol. Struct. 26: 401-24.

Other methods compete with phage display in peptide research. A peptidelibrary can be fused to the carboxyl terminus of the lac repressor andexpressed in E. coli. Another E. coli-based method allows display on thecell's outer membrane by fusion with a peptidoglycan-associatedlipoprotein (PAL). These and related methods are collectively referredto as “E. coli display.” Another biological approach to screeningsoluble peptide mixtures uses yeast for expression and secretion. SeeSmith et al. (1993), Mol. Pharmacol. 43: 741-8. The method of Smith etal. and related methods are referred to as “yeast-based screening.” Inanother method, translation of random RNA is halted prior to ribosomerelease, resulting in a library of polypeptides with their associatedRNA still attached. This and related methods are collectively referredto as “ribosome display.” Other methods employ chemical linkage ofpeptides to RNA; see, for example, Roberts & Szostak (1997), Proc. Natl.Acad. Sci. USA, 94: 12297-303. This and related methods are collectivelyreferred to as “RNA-peptide screening.” Chemically derived peptidelibraries have been developed in which peptides are immobilized onstable, non-biological materials, such as polyethylene rods orsolvent-permeable resins. Another chemically derived peptide libraryuses photolithography to scan peptides immobilized on glass slides.These and related methods are collectively referred to as“chemical-peptide screening.” Chemical-peptide screening may beadvantageous in that it allows use of D-amino acids and other unnaturalanalogues, as well as non-peptide elements. Both biological and chemicalmethods are reviewed in Wells & Lowman (1992), Curr. Opin. Biotechnol.3: 355-62.

In the case of known bioactive peptides, rational design of peptideligands with favorable therapeutic properties can be carried out. Insuch an approach, stepwise changes are made to a peptide sequence andthe effect of the substitution upon bioactivity or a predictivebiophysical property of the peptide (e.g., solution structure) isdetermined. These techniques are collectively referred to as “rationaldesign.” In one such technique, a series of peptides is made in which asingle residue at a time is replaced with alanine. This technique iscommonly referred to as an “alanine walk” or an “alanine scan.” When tworesidues (contiguous or spaced apart) are replaced, it is referred to asa “double alanine walk.” The resultant amino acid substitutions can beused alone or in combination to result in a new peptide entity withfavorable therapeutic properties.

Structural analysis of protein-protein interaction may also be used tosuggest peptides that mimic the binding activity of large proteinligands. In such an analysis, the crystal structure may suggest theidentity and relative orientation of critical residues of the largeprotein ligand, from which a peptide may be designed. See, e.g.,Takasaki et al. (1997), Nature Biotech. 15: 1266-70. These and relatedmethods are referred to as “protein structural analysis.” Theseanalytical methods may also be used to investigate the interactionbetween a receptor protein and peptides selected by phage display, whichmay suggest further modification of the peptides to increase bindingaffinity.

Conceptually, peptide mimetics of any protein can be identified usingphage display and the other methods mentioned above. These methods havealso been used for epitope mapping, for identification of critical aminoacids in protein-protein interactions, and as leads for the discovery ofnew therapeutic agents. E.g., Cortese et al. (1996), Curr. Opin.Biotech. 7: 616-21. Peptide libraries are now being used most often inimmunological studies, such as epitope mapping. Kreeger (1996), TheScientist 10(13): 19-20.

Of particular interest is use of peptide libraries and other techniquesin the discovery of pharmacologically active peptides. A number of suchpeptides identified in the art are summarized in Table 2. The peptidesare described in the listed publications, each of which is herebyincorporated by reference. The pharmacologic activity of the peptides isdescribed, and in many instances is followed by a shorthand termtherefor in parentheses. Some of these peptides have been modified(e.g., to form C-terminally cross-linked dimers). Typically, peptidelibraries were screened for binding to a receptor for apharmacologically active protein (e.g., EPO receptor). In at least oneinstance (CTLA4), the peptide library was screened for binding to amonoclonal antibody.

TABLE 2 Pharmacologically active peptides Binding partner/ Form ofprotein of Pharmacologic peptide interest¹ activity Referenceintrapeptide EPO receptor EPO-mimetic Wrighton et al. (1996), disulfide-Science 273: 458-63; U.S. bonded Pat. No. 5,773,569, issued Jun. 30,1998 to Wrighton et al. C-terminally EPO receptor EPO-mimetic Livnah etal. (1996), cross-linked Science 273: 464-71; dimer Wrighton et al.(1997), Nature Biotechnology 15: 1261-5; International patentapplication WO 96/40772, published Dec. 19, 1996 linear EPO receptorEPO-mimetic Naranda et al. (1999), Proc. Natl. Acad. Sci. USA, 96:7569-74; WO 99/47151, published Sep. 23, 1999 linear c-Mpl TPO-mimeticCwirla et al.(1997) Science 276: 1696-9; U.S. Pat. No. 5,869,451, issuedFeb. 9, 1999; U.S. Pat. No. 5,932,946, issued Aug. 3, 1999 C-terminallyc-Mpl TPO-mimetic Cwirla et al. (1997), cross-linked Science 276: 1696-9dimer disulfide- stimulation of hematopoiesis Paukovits et al. (1984),linked dimer (“G-CSF-mimetic”) Hoppe-Seylers Z. Physiol. Chem. 365:303-11; Laerum et al. (1988), Exp. Hemat. 16: 274-80 alkylene-G-CSF-mimetic Bhatnagar et al. (1996), J. linked dimer Med. Chem. 39:3814-9; Cuthbertson et al. (1997), J. Med. Chem. 40: 2876-82; King etal. (1991), Exp. Hematol. 19: 481; King et al. (1995), Blood 86 (Suppl.1): 309a linear IL-1 receptor inflammatory and U.S. Pat. No. 5,608,035;autoimmune diseases U.S. Pat. No. 5,786,331; (“IL-1 antagonist” or U.S.Pat. No. 5,880,096; “IL-1ra-mimetic”) Yanofsky et al. (1996), Proc.Natl. Acad. Sci. 93: 7381-6; Akeson et al. (1996), J. Biol. Chem. 271:30517-23; Wiekzorek et al. (1997), Pol. J. Pharmacol. 49: 107-17;Yanofsky (1996), PNAs, 93: 7381-7386. linear Facteur thymiquestimulation of lymphocytes Inagaki-Ohara et al. (1996), serique (FTS)(“FTS-mimetic”) Cellular Immunol. 171: 30-40; Yoshida (1984), Int. J.Immunopharmacol, 6: 141-6. intrapeptide CTLA4 MAb CTLA4-mimetic Fukumotoet al. (1998), disulfide Nature Biotech. 16: 267-70 bonded exocyclicTNF-α receptor TNF-α antagonist Takasaki et al. (1997), Nature Biotech.15: 1266-70; WO 98/53842, published Dec. 3, 1998 linear TNF-α receptorTNF-α antagonist Chirinos-Rojas (1998), J. Imm., 5621-5626. intrapeptideC3b inhibition of complement Sahu et al. (1996), J. disulfideactivation; autoimmune Immunol. 157: 884-91; bonded diseases Morikis etal. (1998), (“C3b-antagonist”) Protein Sci. 7: 619-27 linear vinculincell adhesion processes- Adey et al. (1997), cell growth,differentiation, Biochem. J. 324: 523-8 wound healing, tumor metastasis(“vinculin binding”) linear C4 binding anti-thrombotic Linse et al.(1997), J. Biol. protein (C4BP) Chem. 272: 14658-65 linear urokinasereceptor processes associated with Goodson et al. (1994), urokinaseinteraction with its Proc. Natl. Acad. Sci. 91: receptor (e.g.,angiogenesis, 7129-33; International tumor cell invasion and applicationWO 97/35969, metastasis); (“UKR published Oct. 2, 1997 antagonist”)linear Mdm2, Hdm2 Inhibition of inactivation of Picksley et al. (1994),p53 mediated by Mdm2 or Oncogene 9: 2523-9; hdm2; anti-tumor Bottger etal. (1997) J. Mol. (“Mdm/hdm antagonist”) Biol. 269: 744-56; Bottger etal. (1996), Oncogene 13: 2141-7 linear p21^(WAF1) anti-tumor bymimicking the Ball et al. (1997), Curr. activity of p21^(WAF1) Biol. 7:71-80 linear farnesyl anti-cancer by preventing Gibbs et al. (1994),Cell transferase activation of ras oncogene 77: 175-178 linear Raseffector anti-cancer by inhibiting Moodie et al. (1994), domainbiological function of the ras Trends Genet 10: 44-48 oncogene Rodriguezet al. (1994), Nature 370: 527-532 linear SH2/SH3 anti-cancer byinhibiting Pawson et al (1993), Curr. domains tumor growth withactivated Biol. 3: 434-432 tyrosine kinases; treatment Yu et al. (1994),Cell of SH3-mediated disease 76: 933-945; Rickles et al. states (“SH3antagonist”) (1994), EMBO J. 13: 5598-5604; Sparks et al. (1994), J.Biol. Chem. 269: 23853-6; Sparks et al. (1996), Proc. Natl. Acad. Sci.93: 1540-4; U.S. Pat. No. 5,886,150, issued Mar. 23, 1999; U.S. Pat. No.5,888,763, issued Mar. 30, 1999 linear p16^(INK4) anti-cancer bymimicking Fåhraeus et al. (1996), activity of p16; e.g., Curr. Biol. 6:84-91 inhibiting cyclin D-Cdk complex (“p16-mimetic”) linear Src, Lyninhibition of Mast cell Stauffer et al. (1997), activation, IgE-relatedBiochem. 36: 9388-94 conditions, type I hypersensitivity (“Mast cellantagonist”) linear Mast cell protease treatment of inflammatoryInternational application disorders mediated by WO 98/33812, publishedrelease of tryptase-6 Aug. 6, 1998 (“Mast cell protease inhibitors”)linear HBV core antigen treatment of HBV viral Dyson & Muray (1995),(HBcAg) infections (“anti-HBV”) Proc. Natl. Acad. Sci. 92: 2194-8 linearselectins neutrophil adhesion; Martens et al. (1995), J. inflammatorydiseases Biol. Chem. 270: 21129-36; (“selectin antagonist”) Europeanpatent application EP 0 714 912, published Jun. 5, 1996 linear, cyclizedcalmodulin calmodulin antagonist Pierce et al. (1995), Molec. Diversity1: 259-65; Dedman et al. (1993), J. Biol. Chem. 268: 23025-30; Adey &Kay (1996), Gene 169: 133-4 linear, integrins tumor-homing; treatmentfor International applications cyclized- conditions related to WO95/14714, published integrin-mediated cellular Jun. 1, 1995; WO events,including platelet 97/08203, published Mar. aggregation, thrombosis, 6,1997; WO 98/10795, wound healing, published Mar. 19, 1998; osteoporosis,tissue repair, WO 99/24462, published angiogenesis (e.g., for May 20,1999; Kraft et al. treatment of cancer), and (1999), J. Biol. Chem. 274:tumor invasion 1979-1985 (“integrin-binding”) cyclic, linear fibronectinand treatment of inflammatory WO 98/09985, published extracellular andautoimmune conditions Mar. 12, 1998 matrix components of T cells andmacrophages linear somatostatin and treatment or prevention of Europeanpatent application cortistatin hormone-producing tumors, 0 911 393,published Apr. acromegaly, giantism, 28, 1999 dementia, gastric ulcer,tumor growth, inhibition of hormone secretion, modulation of sleep orneural activity linear bacterial antibiotic; septic shock; U.S. Pat. No.5,877,151, lipopolysaccharide disorders modulatable by issued Mar. 2,1999 CAP37 linear or pardaxin, mellitin antipathogenic WO 97/31019,published cyclic, 28 Aug. 1997 including D- amino acids linear, cyclicVIP impotence, WO 97/40070, published neurodegenerative disorders Oct.30, 1997 linear CTLs cancer EP 0 770 624, published May 2, 1997 linearTHF-gamma2 Burnstein (1988), Biochem., 27: 4066-71. linear Amylin Cooper(1987), Proc. Natl. Acad. Sci., 84: 8628-32. linear AdrenomedullinKitamura (1993), BBRC, 192: 553-60. cyclic, linear VEGF anti-angiogenic;cancer, Fairbrother (1998), rheumatoid arthritis, diabetic Biochem., 37:17754-17764. retinopathy, psoriasis (“VEGF antagonist”) cyclic MMPinflammation and Koivunen (1999), Nature autoimmune disorders; Biotech.,17: 768-774. tumor growth (“MMP inhibitor”) HGH fragment treatment ofobesity U.S. Pat. No. 5,869,452 Echistatin inhibition of platelet Gan(1988), J. Biol. Chem., aggregation 263: 19827-32. linear SLEautoantibody SLE WO 96/30057, published Oct. 3, 1996 GD1alphasuppression of tumor Ishikawa et al. (1998), metastasis FEBS Lett. 441(1): 20-4 antiphospholipid endothelial cell activation, Blank et al.(1999), Proc. beta-2- antiphospholipid syndrome Natl. Acad. Sci. USA 96:glycoprotein-I (APS), thromboembolic 5164-8 (β2GPI) phenomena,antibodies thrombocytopenia, and recurrent fetal loss linear T CellReceptor diabetes WO 96/11214, published beta chain Apr. 18, 1996.Antiproliferative, antiviral WO 00/01402, published Jan. 13, 2000.anti-ischemic, growth WO 99/62539, published hormone-liberating Dec. 9,1999. anti-angiogenic WO 99/61476, published Dec. 2, 1999. linearApoptosis agonist; treatment WO 99/38526, published of T cell-associatedAug. 5, 1999. disorders (e.g., autoimmune diseases, viral infection, Tcell leukemia, T cell lymphoma) linear MHC class II treatment ofautoimmune U.S. Pat. No. 5,880,103, diseases issued Mar. 9, 1999. linearandrogen R, p75, proapoptotic, useful in WO 99/45944, published MJD,DCC, treating cancer Sep. 16, 1999. huntingtin linear von Willebrandinhibition of Factor VIII WO 97/41220, published Factor; Factorinteraction; anticoagulants Apr. 29, 1997. VIII linear lentivirus LLP1antimicrobial U.S. Pat. No. 5,945,507, issued Aug. 31, 1999. linearDelta-Sleep sleep disorders Graf (1986), Peptides Inducing Peptide 7:1165. linear C-Reactive inflammation and cancer Barna (1994), CancerProtein (CRP) Immunol. Immunother. 38: 38 (1994). linearSperm-Activating infertility Suzuki (1992), Comp. Peptides Biochem.Physiol. 102B: 679. linear angiotensins hematopoietic factors forLundergan (1999), J. hematocytopenic conditions Periodontal Res. 34(4):223-228. from cancer, AIDS, etc. linear HIV-1 gp41 anti-AIDS Chan(1998), Cell 93: 681-684. linear PKC inhibition of bone resorptionMoonga (1998), Exp. Physiol. 83: 717-725. linear defensins (HNP-antimicrobial Harvig (1994), Methods 1, -2, -3, -4) Enz. 236: 160-172.linear p185^(HER2/neu), C- AHNP-mimetic: anti-tumor Park (2000), Nat.erbB-2 Biotechnol. 18: 194-198. linear gp130 IL-6 antagonist WO99/60013, published Nov. 25, 1999. linear collagen, other autoimmunediseases WO 99/50282, published joint, cartilage, Oct. 7, 1999.arthritis-related proteins linear HIV-1 envelope treatment ofneurological WO 99/51254, published protein degenerative diseases Oct.14, 1999. linear IL-2 autoimmune disorders (e.g., WO 00/04048, publishedgraft rejection, rheumatoid Jan. 27, 2000; WO arthritis) 00/11028,published Mar. 2, 2000. ¹The protein listed in this column may be boundby the associated peptide (e.g., EPO receptor, IL-1 receptor) ormimicked by the associated peptide. The references listed for eachclarify whether the molecule is bound by or mimicked by the peptides.

Peptides identified by peptide library screening have been regarded as“leads” in development of therapeutic agents rather than being used astherapeutic agents themselves. Like other proteins and peptides, theywould be rapidly removed in vivo either by renal filtration, cellularclearance mechanisms in the reticuloendothelial system, or proteolyticdegradation. (Francis (1992), Focus on Growth Factors 3: 4-11.) As aresult, the art presently uses the identified peptides to validate drugtargets or as scaffolds for design of organic compounds that might nothave been as easily or as quickly identified through chemical libraryscreening. Lowman (1997), Ann. Rev. Biophys. Biomol. Struct. 26: 401-24;Kay et al. (1998), Drug Disc. Today 3: 370-8.

Typically, purified peptides are only marginally stable in an aqueousstate and undergo chemical and physical degradation resulting in a lossof biological activity during processing and storage. Additionally,peptide compositions in aqueous solution undergo hydrolysis, such asdeamidation and peptide bond cleavage. These effects represent a seriousproblem for therapeutically active peptides which are intended to beadministered to humans within a defined dosage range based on biologicalactivity.

Administration of purified peptides remains a promising treatmentstrategy for many diseases that affect the human population. However,the ability of the therapeutic peptibody to remain a stablepharmaceutical composition over time in a variety of storage conditionsand then be effective for patients in vivo has not been addressed. Thus,there remains a need in the art to provide therapeutic peptibodies instable formulations that are useful as therapeutic agents for thetreatment of diseases and disorders.

SUMMARY OF THE INVENTION

The present invention provides formulations useful for lyophilization oftherapeutic peptibodies, resulting in a highly stable therapeuticpeptibody product. The stable therapeutic peptibody product is useful asa therapeutic agent in the treatment of individuals suffering fromdisorders or conditions that can benefit from the administration of thetherapeutic peptibody.

In one aspect, the invention provides a lyophilized therapeuticpeptibody composition comprising a buffer, a bulking agent, astabilizing agent, and optionally a surfactant; wherein the buffer iscomprised of a pH buffering agent in a range of about 5 mM to about 20mM and wherein the pH is in a range of about 3.0 to about 8.0; whereinthe bulking agent is at a concentration of about 0% to about 4.5% w/v;wherein the stabilizing agent is at a concentration of about 0.1% toabout 20% w/v; wherein the surfactant is at a concentration of about0.004% to about 0.4% w/v; and wherein the therapeutic peptibodycomprises a structure set out in Formula I,

[(X¹)_(a)—F¹—(X²)_(b)]-(L¹)_(c)-WSP_(d)  Formula I

wherein:

F¹ is an Fc domain;

X¹ is selected from

-   -   P¹-(L²)_(e)—    -   P²-(L³)_(f)-P¹-(L²)_(e)—    -   P³-(L⁴)-P²-(L³)_(f)-P¹-(L²)_(e)- and    -   P⁴-(L⁵)_(h)-P³-(L⁴)_(g)-P²-(L³)_(f)-P¹-(L²)_(e)—

X² is selected from:

-   -   -(L²)_(e)-P¹,    -   -(L²)_(e)-P¹-(L³)_(f)-P²,    -   -(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³, and    -   -(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³-(L⁵)_(h)-P⁴

wherein P¹, P², P³, and P⁴ are each independently sequences ofpharmacologically active peptides;

L¹, L², L³, L⁴, and L⁵ are each independently linkers;

a, b, c, e, f, g, and h are each independently 0 or 1,

-   -   provided that at least one of a and b is 1;

d is 0, 1, or greater than 1; and

WSP is a water soluble polymer, the attachment of which is effected atany reactive moiety in F¹.

In another embodiment, the therapeutic peptibody comprises a structureset out in Formula II

[X¹—F¹]-(L¹)_(c)-WSP_(d)  Formula II

wherein the Fc domain is attached at the C-terminus of X¹, and zero, oneor more WSP is attached to the Fc domain, optionally through linker L¹.

In still another embodiment, the therapeutic peptibody comprises astructure set out in Formula III

[F¹—X²]-(L¹)_(c)-WSP_(d)  Formula III

wherein the Fc domain is attached at the N-terminus of X², and zero, oneor more WSP is attached to the Fc domain, optionally through linker L¹.

In yet another embodiment, the therapeutic peptibody comprises astructure set out in Formula IV

[F¹-(L¹)_(e)-P¹]-(L¹)_(c)-WSP_(d)  Formula IV

wherein the Fc domain is attached at the N-terminus of -(L¹)_(c)-P¹ andzero, one or more WSP is attached to the Fc domain, optionally throughlinker L¹.

In another embodiment, the therapeutic peptibody comprises a structureset out in Formula V

[F¹-(L¹)_(e)-P¹-(L²)_(f)-P²]-(L¹)_(c)-WSP_(d)  Formula V

wherein the Fc domain is attached at the N-terminus of -L¹-P¹-L²-P² andzero, one or more WSP is attached to the Fc domain, optionally throughlinker L¹.

In another embodiment, the therapeutic peptibody is a multimer or dimer.In another embodiment, an aforementioned composition is provided whereinP¹, P², P³ and/or P⁴ are independently selected from a peptide set outin any one of Tables 4 through 38. In a related embodiment, P¹, P², P³and/or P⁴ have the same amino acid sequence. In another embodiment, theFc domain is set out in SEQ ID NO:1. In another embodiment, WSP is PEG.In still another embodiment, the Fc domain is et out in SEQ ID NO:1 andWSP is PEG. In another embodiment, the PEG has a molecular weight ofbetween about 2 kDa and 100 kDa, or between 6 kDa and 25 kDa. In anotherembodiment, the composition comprises at least 50%, 75%, 85%, 90%, or95% PEGylated therapeutic peptibody.

In yet another embodiment of the invention, an aforementionedcomposition is provided wherein the pH buffering agent is selected fromthe group consisting of glycine, histidine, glutamate, succinate,phosphate, acetate, and aspartate. In yet another embodiment of theinvention, an aforementioned composition is provided wherein the bulkingagent selected from the group consisting of mannitol, glycine, sucrose,dextran, polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol,trehalose, or xylitol.

In yet another embodiment of the invention, an aforementionedcomposition is provided wherein the stabilizing agent selected from thegroup consisting of sucrose, trehalose, mannose, maltose, lactose,glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose,glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL,poly-hydroxy compounds, including polysaccharides such as dextran,starch, hydroxyethyl starch, cyclodextrins, N-methylpyrollidene,cellulose and hyaluronic acid, sodium chloride.

In yet another embodiment of the invention, an aforementionedcomposition is provided wherein the surfactant selected from the groupconsisting of sodium lauryl sulfate, dioctyl sodium sulfosuccinate,dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosinesodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt,sodium cholate hydrate, sodium deoxycholate, glycodeoxycholic acidsodium salt, benzalkonium chloride or benzethonium chloride,cetylpyridinium chloride monohydrate, hexadecyltrimethylammoniumbromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, TritonX-114, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylenehydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate,polysorbate 20, 40, 60, 65 and 80, soy lecithin, DOPC, DMPG, DMPC, andDOPG; sucrose fatty acid ester, methyl cellulose and carboxymethylcellulose. In yet another embodiment of the invention, an aforementionedcomposition is provided wherein the therapeutic peptibody concentrationis between about 0.25 mg/mL and 250 mg/mL.

In another embodiment of the invention, an aforementioned composition isprovided wherein the pH buffering agent is 10 mM histidine and whereinthe pH is 5.0; wherein the bulking agent is 4% w/v mannitol; wherein thestabilizing agent is 2% w/v sucrose; and wherein the surfactant is0.004% w/v polysorbate-20. In another embodiment, the aforementionedcomposition is provided wherein P¹ comprises a sequence set forth inTable 6. In yet another embodiment of the invention, an aforementionedcomposition is provided wherein the therapeutic peptibody concentrationis 0.5 mg/mL. In another embodiment, the therapeutic peptibody is anyone of SEQ ID NO:993, SEQ ID NO:994, SEQ ID NO:995, SEQ ID NO:996, SEQID NO:997, SEQ ID NO:998, SEQ ID NO:999, SEQ ID NO:1000, SEQ ID NO:1001,SEQ ID NO:1002, SEQ ID NO:1003, SEQ ID NO:1004, SEQ ID NO:1005, SEQ IDNO:1006, SEQ ID NO:1007, SEQ ID NO:1008, SEQ ID NO:1009, SEQ ID NO:1010,SEQ ID NO:1011, SEQ ID NO:1012, SEQ ID NO:1013, SEQ ID NO:1014, SEQ IDNO:1015, SEQ ID NO:1016, or SEQ ID NO:1017.

In yet another embodiment of the invention, an aforementionedcomposition is provided wherein the pH buffering agent is 10 mMhistidine and wherein the pH is 7.0; wherein the bulking agent is 4% w/vmannitol; wherein the stabilizing agent is 2% w/v sucrose; and whereinthe surfactant is 0.01% w/v polysorbate-20. In another embodiment, theaforementioned composition is provided wherein P¹ comprises a sequenceset forth in Table 32. In yet another embodiment of the invention, anaforementioned composition is provided wherein the therapeutic peptibodyconcentration is 30 mg/mL.

In still another embodiment of the invention, an aforementionedcomposition is provided wherein the pH buffering agent is 20 mMhistidine and wherein the pH is 5.0; wherein the bulking agent is 3.3%w/v mannitol; wherein the stabilizing agent is 2% w/v sucrose; andwherein the surfactant is 0.01% w/v polysorbate-20. In anotherembodiment, the aforementioned composition is provided wherein P¹comprises a sequence set forth in Table 4. In yet another embodiment ofthe invention, an aforementioned composition is provided wherein thetherapeutic peptibody concentration is 100 mg/mL.

In still another embodiment of the invention, an aforementionedcomposition is provided wherein the pH buffering agent is 10 mMhistidine and wherein the pH is 5.0; wherein the bulking agent is 2.5%w/v mannitol; and wherein the stabilizing agent is 3.5% w/v sucrose. Inanother embodiment, the aforementioned composition is provided whereinP¹ comprises a sequence set forth in Table 31. In yet another embodimentof the invention, an aforementioned composition is provided wherein thetherapeutic peptibody concentration is 30 mg/mL.

In another embodiment of the invention, an aforementioned composition isprovided wherein the composition is selected from the group consistingof: a) 10 mM histidine, pH 4.7, 4% mannitol and 2% sucrose, with andwithout 0.004% polysorbate-20; b) 10 mM histidine, pH 5, 4% mannitol and2% sucrose, with and without 0.004% polysorbate-20; c) 10 mM glutamate,pH 4.5, 4% mannitol and 2% sucrose with and without 0.004%polysorbate-20; d) 10 mM succinate, pH 4.5, 4% mannitol and 2% sucrose,0.004% polysorbate-20; e) 10 mM glutamate, pH 4.5, 9% sucrose, 0.004%polysorbate-20; f) 10 mM glutamate, pH 4.5, 4% mannitol, 2% sucrose, 1%hydroxyethyl starch, 0.004% polysorbate-20; g) 5 mM glutamate, pH 4.5,2% mannitol, 1% sucrose, 0.004% polysorbate-20; and h) 10 mM glutamate,pH 4.5, 4% mannitol, 2% trehalose, 0.004% polysorbate-20. In anotherembodiment, the aforementioned composition is provided wherein P¹comprises a sequence set forth in Tables 21-24. In still anotherembodiment, the aforementioned composition is provided wherein thetherapeutic peptibody concentration is selected from the groupconsisting of 1, 30, 85, and 100 mg/mL.

The present invention also contemplates methods of making lyophilizedtherapeutic peptibodies of the present invention. In one embodiment, theinvention provides a method for making a lyophilized therapeuticpeptibody comprising the steps of: a) preparing a solution of a buffer,a bulking agent, a stabilizing agent, and a optionally surfactant;wherein the buffer is comprised of a pH buffering agent in a range ofabout 5 mM to about 20 mM and wherein the pH is in a range of about 3.0to about 8.0; wherein the bulking agent is at a concentration of about2.5% to about 4% w/v; wherein the stabilizing agent is at aconcentration of about 0.1% to about 5% w/v; wherein the surfactant isat a concentration of about 0.004% to about 0.04% w/v; and b)lyophilizing the therapeutic peptibody; wherein the therapeuticpeptibody comprises a structure set out in Formula I,

[(X¹)_(a)—F¹—(X²)_(b)]-(L¹)_(c)-WSP_(d)  Formula I

wherein:

F¹ is an Fc domain;

X¹ is selected from

-   -   P¹-(L²)_(e)—    -   P²-(L³)_(f)-P¹-(L²)_(e)—    -   P³-(L⁴)_(g)-P²-(L³)_(f)-P¹-(L²)_(e)- and    -   P⁴-(L⁵)_(h)-P³-(L⁴)_(g)-P²-(L³)_(f)-P¹-(L²)_(e)

X² is selected from:

-   -   -(L²)_(e)-P¹,    -   -(L²)_(e)-P¹-(L¹)_(f)-P²,    -   -(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³, and    -   -(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³-(L⁵)_(h)-P⁴

wherein P¹, P², P³, and P⁴ are each independently sequences ofpharmacologically active peptides;

L¹, L², L³, L⁴, and L⁵ are each independently linkers;

a, b, c, e, f, g, and h are each independently 0 or 1,

-   -   provided that at least one of a and b is 1;

d is 0, 1, or greater than 1; and

WSP is a water soluble polymer, the attachment of which is effected atany reactive moiety in F¹.

In another embodiment, the aforementioned method is provided wherein thetherapeutic peptibody comprises a structure set out in Formula II

[X¹—F¹]-(L¹)_(c)-WSP_(d)  Formula II

wherein the Fc domain is attached at the C-terminus of X¹, and zero, oneor more WSP is attached to the Fc domain, optionally through linker L¹.

In another embodiment, the aforementioned method is provided wherein thetherapeutic peptibody comprises a structure set out in Formula III

[F¹—X²]-(L¹)_(c)-WSP_(d)  Formula III

wherein the Fc domain is attached at the N-terminus of X², and zero, oneor more WSP is attached to the Fc domain, optionally through linker L¹.

In another embodiment, the aforementioned method is provided wherein thetherapeutic peptibody comprises a structure set out in Formula IV

[F¹-(L¹)_(e)-P¹]-(L¹)_(c)-WSP_(d)  Formula IV

wherein the Fc domain is attached at the N-terminus of -(L¹)_(c)-P¹ andzero, one or more WSP is attached to the Fc domain, optionally throughlinker L¹.

In another embodiment, the aforementioned method is provided wherein thetherapeutic peptibody comprises a structure set out in Formula V

[F¹-(L¹)_(e)-P¹-(L²)_(f)-P²]-(L¹)_(c)-WSP_(d)  Formula V

wherein the Fc domain is attached at the N-terminus of -L¹-P¹-L²-P² andzero, one or more WSP is attached to the Fc domain, optionally throughlinker L¹.

In another embodiment, the aforementioned method is provided wherein thetherapeutic peptibody is a multimer or dimer. In another embodiment, theP¹, P², P³ and/or P⁴ are independently selected from a peptide set outin any one of Tables 4 through 38. In another embodiment, the P¹, P², P³and/or P⁴ have the same amino acid sequence. In another embodiment, theFc domain is set out in SEQ ID NO:1. In another embodiment, WSP is PEG.In another embodiment, the Fc domain is set out in SEQ ID NO:1 and WSPis PEG. In another embodiment, PEG has a molecular weight of betweenabout 2 kDa and 100 kDa or between about 6 kDa and 25 kDa. In anotherembodiment, the aforementioned method is provided wherein thecomposition comprises at least 50%, 75%, 85%, 90%, or 95% PEGylatedtherapeutic peptibody.

In another embodiment, the aforementioned method is provided wherein thepH buffering agent is selected from the group consisting of glycine,histidine, glutamate, succinate, phosphate, acetate, and aspartate. Inanother embodiment, the aforementioned method is provided wherein thebulking agent selected from the group consisting of mannitol, glycine,sucrose, dextran, polyvinylpyrolidone, carboxymethylcellulose, lactose,sorbitol, trehalose, or xylitol. In another embodiment, theaforementioned method is provided wherein the stabilizing agent selectedfrom the group consisting of sucrose, trehalose, mannose, maltose,lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose,arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginineHCL, poly-hydroxy compounds, including polysaccharides such as dextran,starch, hydroxyethyl starch, cyclodextrins, N-methylpyrollidene,cellulose and hyaluronic acid, sodium chloride.

In another embodiment, the aforementioned method is provided wherein thesurfactant selected from the group consisting of sodium lauryl sulfate,dioctyl sodium sulfosuccinate, dioctyl sodium sulfonate,chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecylsulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate,sodium deoxycholate, glycodeoxycholic acid sodium salt, benzalkoniumchloride or benzethonium chloride, cetylpyridinium chloride monohydrate,hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12,digitonin, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60,glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, soy lecithin,DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methyl celluloseand carboxymethyl cellulose. In another embodiment, the aforementionedmethod is provided wherein the therapeutic peptibody concentration isbetween about 0.25 mg/mL and 250 mg/mL.

In another embodiment, an aforementioned method is provided wherein thepH buffering agent is 10 mM histidine and wherein the pH is 5.0; whereinthe bulking agent is 4% w/v mannitol; wherein the stabilizing agent is2% w/v sucrose; and wherein the surfactant is 0.004% w/v polysorbate-20.In another embodiment, the aforementioned method is provided wherein P¹comprises a sequence set forth in Table 6. In another embodiment, theaforementioned method is provided wherein the therapeutic peptibodyconcentration is 0.5 mg/mL.

In another embodiment, an aforementioned method is provided wherein thepH buffering agent is 10 mM histidine and wherein the pH is 7.0; whereinthe bulking agent is 4% w/v mannitol; wherein the stabilizing agent is2% w/v sucrose; and wherein the surfactant is 0.01% w/v polysorbate-20.In another embodiment, the aforementioned method is provided wherein P¹comprises a sequence set forth in Table 32. In another embodiment, theaforementioned method is provided wherein the therapeutic peptibodyconcentration is 30 mg/mL.

In another embodiment, an aforementioned method is provided wherein thepH buffering agent is 20 mM histidine and wherein the pH is 5.0; whereinthe bulking agent is 3.3% w/v mannitol; wherein the stabilizing agent is2% w/v sucrose; and wherein the surfactant is 0.01% w/v polysorbate-20.In another embodiment, the aforementioned method is provided wherein P¹comprises a sequence set forth in Table 4. In another embodiment, theaforementioned method is provided wherein the therapeutic peptibodyconcentration is 100 mg/mL.

In another embodiment, an aforementioned method is provided wherein thepH buffering agent is 10 mM histidine and wherein the pH is 5.0; whereinthe bulking agent is 2.5% w/v mannitol; and wherein the stabilizingagent is 3.5% w/v sucrose. In another embodiment, the aforementionedmethod is provided wherein P¹ comprises a sequence set forth in Table31. In another embodiment, the aforementioned method is provided whereinthe therapeutic peptibody concentration is 30 mg/mL.

In another embodiment of the invention, an aforementioned method isprovided wherein the composition is selected from the group consistingof: a) 10 mM histidine, pH 4.7, 4% mannitol and 2% sucrose, with andwithout 0.004% polysorbate-20; b) 10 mM histidine, pH 5, 4% mannitol and2% sucrose, with and without 0.004% polysorbate-20; c) 10 mM glutamate,pH 4.5, 4% mannitol and 2% sucrose with and without 0.004%polysorbate-20; d) 10 mM succinate, pH 4.5, 4% mannitol and 2% sucrose,0.004% polysorbate-20; e) 10 mM glutamate, pH 4.5, 9% sucrose, 0.004%polysorbate-20; f) 10 mM glutamate, pH 4.5, 4% mannitol, 2% sucrose, 1%hydroxyethyl starch, 0.004% polysorbate-20; g) 5 mM glutamate, pH 4.5,2% mannitol, 1% sucrose, 0.004% polysorbate-20; and h) 10 mM glutamate,pH 4.5, 4% mannitol, 2% trehalose, 0.004% polysorbate-20. In anotherembodiment, the aforementioned method is provided wherein P¹ comprises asequence set forth in Tables 21-24. In still another embodiment, theaforementioned method is provided wherein the therapeutic peptibodyconcentration is selected from the group consisting of 1, 30, 85, and100 mg/mL.

In another embodiment, an aforementioned method is provided furthercomprising, prior to lyophilization, the steps of: b) adjusting the pHof the solution to a pH between about 4.0 and about 8.0; c) preparing asolution containing the therapeutic peptibody; d) buffer exchanging thesolution of step (c) into the solution of step (b); e) adding anappropriate amount of a surfactant; and f) lyophilizing the mixture fromstep (e).

In another embodiment, the aforementioned method is provided wherein amethod for preparing a reconstituted therapeutic peptibody compositionis provided comprising the steps of: a) lyophilizing an aforementionedtherapeutic peptibody composition; and b) reconstituting the lyophilizedtherapeutic peptibody composition.

In another embodiment, a kit for preparing an aqueous pharmaceuticalcomposition is provided comprising a first container having anaforementioned lyophilized therapeutic peptibody composition, and asecond container having a physiologically acceptable solvent for thelyophilized composition.

DETAILED DESCRIPTION OF THE INVENTION Definition of Terms

The term “comprising,” with respect to a peptide compound, means that acompound may include additional amino acids on either or both of theamino or carboxy termini of the given sequence. Of course, theseadditional amino acids should not significantly interfere with theactivity of the compound. With respect to a composition of the instantinvention, the term “comprising” means that a composition may includeadditional components. These additional components should notsignificantly interfere with the activity of the composition.

The term “peptibody” refers to a molecule comprising peptide(s) fusedeither directly or indirectly to other molecules such as an Fc domain ofan antibody, where the peptide moiety specifically binds to a desiredtarget. The peptide(s) may be fused to either an Fc region or insertedinto an Fc-Loop, a modified Fc molecule. Fc-Loops are described in U.S.Patent Application Publication No. US2006/0140934 incorporated herein byreference in its entirety. The invention includes such moleculescomprising an Fc domain modified to comprise a peptide as an internalsequence (preferably in a loop region) of the Fc domain. The Fc internalpeptide molecules may include more than one peptide sequence in tandemin a particular internal region, and they may include further peptidesin other internal regions. While the putative loop regions areexemplified, insertions in any other non-terminal domains of the Fc arealso considered part of this invention. The term “peptibody” does notinclude Fc-fusion proteins (e.g., full length proteins fused to an Fcdomain).

The term “vehicle” refers to a molecule that prevents degradation and/orincreases half-life, reduces toxicity, reduces immunogenicity, orincreases biological activity of a therapeutic protein. Exemplaryvehicles include an Fc domain as described in U.S. Pat. No. 5,428,130 toCapon et al., issued Jun. 27, 1995.

The term “native Fc” refers to molecule or sequence comprising thesequence of a non-antigen-binding fragment resulting from digestion ofwhole antibody, whether in monomeric or multimeric form. Typically, anative Fc comprises a CH2 and CH3 domain. The original immunoglobulinsource of the native Fc is in one aspect of human origin and may be anyof the immunoglobulins. A native Fc is a monomeric polypeptide that maybe linked into dimeric or multimeric forms by covalent association(i.e., disulficle bonds), non-covalent association or a combination ofboth. The number of intermolecular disulfide bonds between monomericsubunits of native Fc molecules ranges from one to four depending onclass (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1,IgGA2). One example of a native Fc is a disulfide-bonded dimer resultingfrom papain digestion of an IgG. Ellison et al. (1982), Nucleic AcidsRes. 10: 4071-9. The term “native Fc” as used herein is generic to themonomeric, dimeric, and multimeric forms.

The term “Fc variant” refers to a molecule or sequence that is modifiedfrom a native Fc, but still comprises a binding site for the salvagereceptor, FcRn. International applications WO 97/34631 (published 25Sep. 1997) and WO 96/32478 describe exemplary Fc variants, as well asinteraction with the salvage receptor, and are hereby incorporated byreference. In one aspect, the term “Fc variant” comprises a molecule orsequence that is humanized from a non-human native Fc. In anotheraspect, a native Fc comprises sites that may be removed because theyprovide structural features or biological activity that are not requiredfor the fusion molecules of the present invention. Thus, the term “Fcvariant” comprises a molecule or sequence that lacks one or more nativeFc sites or residues that affect or are involved in (1) disulfide bondformation, (2) incompatibility with a selected host cell (3) N-terminalheterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC). Fc variants are described in furtherdetail hereinafter.

The term “Fc domain” encompasses native Fc and Fc variant molecules andsequences as defined above. As with Fc variants and native Fcs, the term“Fc domain” includes molecules in monomeric or multimeric form, whetherdigested from whole antibody or produced by other means. In oneembodiment, for example, the Fc region can be:

(SEQ ID NO: 1) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Additional Fc sequences are known in the art and are contemplated foruse in the invention. For example, Fc IgG1 (GenBank Accession No.P01857), Fc IgG2 (GenBank Accession No. P01859), Fc IgG3 (GenBankAccession No. P01860), Fc IgG4 (GenBank Accession No. P01861), Fc IgA1(GenBank Accession No. P01876), Fc IgA2 (GenBank Accession No. P01877),Fc IgD (GenBank Accession No. P01880), Fc IgM (GenBank Accession No.P01871), and Fc IgE (GenBank Accession No. P01854) are some additionalFc sequences contemplated for use herein.

Optionally, an N-terminal amino acid sequence may be added to the abovesequences (e.g., where expressed in bacteria).

The term “multimer” as applied to Fc domains or molecules comprising Fcdomains refers to molecules having two or more polypeptide chainsassociated covalently, noncovalently, or by both covalent andnon-covalent interactions. IgG molecules typically form dimers; IgM,pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, ortetramers. Multimers may be formed by exploiting the sequence andresulting activity of the native Ig source of the Fc or by derivatizing(as defined below) such a native Fc.

The terms “derivatizing,” “derivative” or “derivatized” mean processesand resulting compounds in which, for example and without limitation,(1) the compound has a cyclic portion; for example, cross-linkingbetween cysteinyl residues within the compound; (2) the compound iscross-linked or has a cross-linking site; for example, the compound hasa cysteinyl residue and thus forms cross-linked dimers in culture or invivo; (3) one or more peptidyl linkage is replaced by a non-peptidyllinkage; (4) the N-terminus is replaced by —NRR₁, NRC(O)R₁, —NRC(O)OR₁,—NRS(O)₂R₁, —NHC(O)NHR, a succinimide group, or substituted orunsubstituted benzyloxycarbonyl-NH—, wherein R and R₁ and the ringsubstituents are as defined hereinafter; (5) the C-terminus is replacedby —C(O)R₂ or —NR₃R₄ wherein R₂, R₃ and R₄ are as defined hereinafter;and (6) compounds in which individual amino acid moieties are modifiedthrough treatment with agents capable of reacting with selected sidechains or terminal residues. Derivatives are further describedhereinafter.

As used herein the term “peptide” refers to molecules of 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100 or more amino acids linked by peptide bonds. Peptidestypically contain random and/or flexible conformations, such as randomcoils; and typically lack stable conformations, such as those observedin larger proteins/polypeptides, typically via secondary and tertiarystructures. In particular embodiments, numerous size ranges of peptidesare contemplated herein, such from about: 3-90, 3-80, 3-70, 3-60, 3-50;5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30; 10-90, 10-80, 10-70, 10-60,10-50, 10-40, 10-30; 10-20 amino acids in length, and the like. Infurther embodiments, the peptides used herein are no more than 100, 90,80, 70, 60, 50, 40, 30, or 20 amino acids in length. Exemplary peptidesmay be generated by any of the methods set forth herein, such as carriedin a peptide library (e.g., a phage display library), generated bychemical synthesis, derived by digestion of proteins, or generated usingrecombinant DNA techniques. Peptides include D and L form, eitherpurified or in a mixture of the two forms.

Additionally, physiologically acceptable salts of the compounds of thisinvention are also contemplated. By “physiologically acceptable salts”is meant any salts that are known or later discovered to bepharmaceutically acceptable. Some specific examples are: acetate;trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide;sulfate; citrate; tartrate; glycolate; and oxalate.

The term “randomized” as used to refer to peptide sequences refers tofully random sequences (e.g., selected by phage display methods) andsequences in which one or more residues of a naturally occurringmolecule is replaced by an amino acid residue not appearing in thatposition in the naturally occurring molecule. Exemplary methods foridentifying peptide sequences include phage display, E. coli display,ribosome display, yeast-based screening, RNA-peptide screening, chemicalscreening, rational design, protein structural analysis, and the like.

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter (e.g.,but not limited to blood pressure, blood cell count, cholesterol level)or disease state (e.g., but not limited to cancer, autoimmunedisorders). Thus, pharmacologically active peptides comprise agonisticor mimetic and antagonistic peptides as defined below.

The terms “-mimetic peptide” and “-agonist peptide” refer to a peptidehaving biological activity comparable to a protein (e.g., but notlimited to EPO, TPO, G-CSF and other proteins described herein) thatinteracts with a protein of interest. These terms further includepeptides that indirectly mimic the activity of a protein of interest,such as by potentiating the effects of the natural ligand of the proteinof interest; see, for example, the G-CSF-mimetic peptides listed inTables 2 and 7. As an example, the term “EPO-mimetic peptide” comprisesany peptides that can be identified or derived as described in Wrightonet al. (1996), Science 273: 458-63, Naranda et al. (1999), Proc. Natl.Acad. Sci. USA 96: 7569-74, or any other reference in Table 2 identifiedas having EPO-mimetic subject matter. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

As another example, the term “TPO-mimetic peptide” or “TMP” refers topeptides that can be identified or derived as described in Cwirla et al.(1997), Science 276: 1696-9, U.S. Pat. Nos. 5,869,451 and 5,932,946 andany other reference in Table 2 identified as having TPO-mimetic subjectmatter, as well as International application WO 00/24770 published May4, 2000, hereby incorporated by reference. Those of ordinary skill inthe art appreciate that each of these references enables one to selectdifferent peptides than actually disclosed therein by following thedisclosed procedures with different peptide libraries.

As another example, the term “G-CSF-mimetic peptide” refers to anypeptides that can be identified or described in Paukovits et al. (1984),Hoppe-Seylers Z. Physiol. Chem. 365: 303-11 or any of the references inTable 2 identified as having G-CSF-mimetic subject matter. Those ofordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The term “CTLA4-mimetic peptide” refers to any peptides that can beidentified or derived as described in Fukumoto et al. (1998), NatureBiotech. 16: 267-70. Those of ordinary skill in the art appreciate thateach of these references enables one to select different peptides thanactually disclosed therein by following the disclosed procedures withdifferent peptide libraries.

The term “-antagonist peptide” or “inhibitor peptide” refers to apeptide that blocks or in some way interferes with the biologicalactivity of the associated protein of interest, or has biologicalactivity comparable to a known antagonist or inhibitor of the associatedprotein of interest. Thus, the term “TNF-antagonist peptide” comprisespeptides that can be identified or derived as described in Takasaki etal. (1997), Nature Biotech. 15: 1266-70 or any of the references inTable 2 identified as having TNF-antagonistic subject matter. Those ofordinary skill in the art appreciate that each of these referencesenables one to select different peptides than actually disclosed thereinby following the disclosed procedures with different peptide libraries.

The terms “IL-1 antagonist” and “IL-1ra-mimetic peptide” refers topeptides that inhibit or down-regulate activation of the IL-1 receptorby IL-1. IL-1 receptor activation results from formation of a complexamong IL-1, IL-1 receptor, and IL-1 receptor accessory protein. IL-1antagonist or IL-1ra-mimetic peptides bind to IL-1, IL-1 receptor, orIL-1 receptor accessory protein and obstruct complex formation among anytwo or three components of the complex. Exemplary IL-1 antagonist orIL-1ra-mimetic peptides can be identified or derived as described inU.S. Pat. Nos. 5,608,035; 5,786,331, 5,880,096; or any of the referencesin Table 2 identified as having IL-1ra-mimetic or IL-1 antagonisticsubject matter. Those of ordinary skill in the art appreciate that eachof these references enables one to select different peptides thanactually disclosed therein by following the disclosed procedures withdifferent peptide libraries.

The term “VEGF-antagonist peptide” refers to peptides that can beidentified or derived as described in Fairbrother (1998), Biochem. 37:17754-64, and in any of the references in Table 2 identified as havingVEGF-antagonistic subject matter. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

The term “MMP inhibitor peptide” refers to peptides that can beidentified or derived as described in Koivunen (1999), Nature Biotech.17: 768-74 and in any of the references in Table 2 identified as havingMMP inhibitory subject matter. Those of ordinary skill in the artappreciate that each of these references enables one to select differentpeptides than actually disclosed therein by following the disclosedprocedures with different peptide libraries.

The term “myostatin inhibitor peptide” refers to peptides that can beidentified by their ability to reduce or block myostatin activity orsignaling as demonstrated in in vitro assays such as, for example thepMARE C2C12 cell-based myostatin activity assay or by in vivo animaltesting as described in U.S. patent application Publication NoUS20040181033A1 and PCT application publication No. WO2004/058988.Exemplary myostatin inhibitor peptides are set out in Tables 21-24.

The term “integrin/adhesion antagonist” refers to peptides that inhibitor down-regulate the activity of integrins, selectins, cell adhesionmolecules, integrin receptors, selectin receptors, or cell adhesionmolecule receptors. Exemplary integrin/adhesion antagonists compriselaminin, echistatin, the peptides described in Tables 25-28.

The term “bone resorption inhibitor” refers to such molecules asdetermined by the assays of Examples 4 and 11 of WO 97/23614: which ishereby incorporated by reference in its entirety. Exemplary boneresorption inhibitors include OPG and OPG-L antibody, which aredescribed in WO 97/23614 and WO98/46751, respectively, which are herebyincorporated by reference in their entirety.

The term “nerve growth factor inhibitor” or “nerve growth factoragonist” refers to a peptide that binds to and inhibits nerve growthfactor (NGF) activity or signaling. Exemplary peptides of this type areset out in Table 29.

The term “TALL-1 modulating domain” refers to any amino acid sequencethat binds to the TALL-1 and comprises naturally occurring sequences orrandomized sequences. Exemplary TALL-1 modulating domains can beidentified or derived by phage display or other methods mentionedherein. Exemplary peptides of this type are set out in Tables 30 and 31.

The term “TALL-1 antagonist” refers to a molecule that binds to theTALL-1 and increases or decreases one or more assay parameters oppositefrom the effect on those parameters by full length native TALL-1. Suchactivity can be determined, for example, by such assays as described inthe subsection entitled “Biological activity of AGP-3” in the Materials& Methods section of the patent application entitled, “TNF-RELATEDPROTEINS”, WO 00/47740, published Aug. 17, 2000.

The term “Ang 2-antagonist peptide” refers to peptides that can beidentified or derived as having Ang-2-antagonistic characteristics.Exemplary peptides of this type are set out in Tables 32-38.

The term “WSP” refers to a water soluble polymer which prevents apeptide, protein or other compound to which it is attached fromprecipitating in an aqueous environment, such as, by way of example, aphysiological environment. A more detailed description of various WSPembodiments contemplated by the invention follows.

Lyophilization and Administration

Therapeutic peptibodies are useful in pharmaceutical formulations inorder to treat human diseases as described herein. In one embodiment thetherapeutic peptibody compositions are lyophilized. Lyophilization iscarried out using techniques common in the art and should be optimizedfor the composition being developed [Tang et al., Pharm Res. 21:191-200,(2004) and Chang et al., Pharm Res. 13:243-9 (1996)].

A lyophilization cycle is, in one aspect, composed of three steps:freezing, primary drying, and secondary drying [A. P. Mackenzie, PhilTrans R Soc London, Ser B, Biol 278:167 (1977)]. In the freezing step,the solution is cooled to initiate ice formation. Furthermore, this stepinduces the crystallization of the bulking agent. The ice sublimes inthe primary drying stage, which is conducted by reducing chamberpressure below the vapor pressure of the ice, using a vacuum andintroducing heat to promote sublimation. Finally, adsorbed or boundwater is removed at the secondary drying stage under reduced chamberpressure and at an elevated shelf temperature. The process produces amaterial known as a lyophilized cake. Thereafter the cake can bereconstituted with either sterile water or suitable diluent forinjection.

The lyophilization cycle not only determines the final physical state ofthe excipients but also affects other parameters such as reconstitutiontime, appearance, stability and final moisture content. The compositionstructure in the frozen state proceeds through several transitions(e.g., glass transitions, wettings, and crystallizations) that occur atspecific temperatures and can be used to understand and optimize thelyophilization process. The glass transition temperature (Tg and/or Tg′)can provide information about the physical state of a solute and can bedetermined by differential scanning calorimetry (DSC). Tg and Tg′ are animportant parameter that must be taken-into account when designing thelyophilization cycle. For example, Tg′ is important for primary drying.Furthermore, in the dried state, the glass transition temperatureprovides information on the storage temperature of the final product.

Excipients in General

Excipients are additives that are included in a formulation because theyeither impart or enhance the stability, delivery and manufacturabilityof a drug product. Regardless of the reason for their inclusion,excipients are an integral component of a drug product and thereforeneed to be safe and well tolerated by patients. For protein drugs, thechoice of excipients is particularly important because they can affectboth efficacy and immunogenicity of the drug. Hence, proteinformulations need to be developed with appropriate selection ofexcipients that afford suitable stability, safety, and marketability.

A lyophilized formulation is usually comprised of a buffer, a bulkingagent, and a stabilizer. The utility of a surfactant may be evaluatedand selected in cases where aggregation during the lyophilization stepor during reconstitution becomes an issue. An appropriate bufferingagent is included to maintain the formulation within stable zones of pHduring lyophilization. A comparison of the excipient components inliquid and lyophilized protein formulations is provided in Table A.

TABLE A Excipient components of lyophilized protein formulationsFunction in lyophilized Excipient component formulation Buffer MaintainpH of formulation during lyophilization and upon reconstitution Tonicityagent/stabilizer Stabilizers include cryo and lyoprotectants Examplesinclude Polyols, sugars and polymers Cryoprotectants protect proteinsfrom freezing stresses Lyoprotectants stabilize proteins in thefreeze-dried state Bulking agent Used to enhance product elegance and toprevent blowout Provides structural strength to the lyo cake Examplesinclude mannitol and glycine Surfactant Employed if aggregation duringthe lyophilization process is an issue May serve to reducereconstitution times Examples include polysorbate 20 and 80 Anti-oxidantUsually not employed, molecular reactions in the lyo cake are greatlyretarded Metal ions/chelating agent May be included if a specific metalion is included only as a co- factor or where the metal is required forprotease activity Chelating agents are generally not needed in lyoformulations Preservative For multi-dose formulations only Providesprotection against microbial growth in formulation Is usually includedin the reconstitution diluent (e.g. bWFI)

The principal challenge in developing formulations for therapeuticproteins is stabilizing the product against the stresses ofmanufacturing, shipping and storage. The role of formulation excipientsis to provide stabilization against these stresses. Excipients may alsobe employed to reduce viscosity of high concentration proteinformulations in order to enable their delivery and enhance patientconvenience. In general, excipients can be classified on the basis ofthe mechanisms by which they stabilize proteins against various chemicaland physical stresses. Some excipients are used to alleviate the effectsof a specific stress or to regulate a particular susceptibility of aspecific protein. Other excipients have more general effects on thephysical and covalent stabilities of proteins. The excipients describedherein are organized either by their chemical type or their functionalrole in formulations. Brief descriptions of the modes of stabilizationare provided when discussing each excipient type.

Given the teachings and guidance provided herein, those skilled in theart will know what amount or range of excipient can be included in anyparticular formulation to achieve a biopharmaceutical formulation of theinvention that promotes retention in stability of the biopharmaceutical.For example, the amount and type of a salt to be included in abiopharmaceutical formulation of the invention can be selected based onto the desired osmolality (i.e., isotonic, hypotonic or hypertonic) ofthe final solution as well as the amounts and osmolality of othercomponents to be included in the formulation. Similarly, byexemplification with reference to the type of polyol or sugar includedin a formulation, the amount of such an excipient will depend on itsosmolality.

By way of example, inclusion of about 5% sorbitol can achieveisotonicity while about 9% of a sucrose excipient is needed to achieveisotonicity. Selection of the amount or range of concentrations of oneor more excipients that can be included within a biopharmaceuticalformulation of the invention has been exemplified above by reference tosalts, polyols and sugars. However, those skilled in the art willunderstand that the considerations described herein and furtherexemplified by reference to specific excipients are equally applicableto all types and combinations of excipients including, for example,salts, amino acids, other tonicity agents, surfactants, stabilizers,bulking agents, cryoprotectants, lyoprotectants, anti-oxidants, metalions, chelating agents and/or preservatives.

Further, where a particular excipient is reported in a formulation by,e.g., percent (%) w/v, those skilled in the art will recognize that theequivalent molar concentration of that excipient is also contemplated.

Of course, a person having ordinary skill in the art would recognizethat the concentrations of the aforementioned excipients share aninterdependency within a particular formulation. By way of example, theconcentration of a bulking agent may be lowered where, e.g., there is ahigh protein/peptide concentration or where, e.g., there is a highstabilizing agent concentration. In addition, a person having ordinaryskill in the art would recognize that, in order to maintain theisotonicity of a particular formulation in which there is no bulkingagent, the concentration of a stabilizing agent would be adjustedaccordingly (i.e., a “tonicifying” amount of stabilizer would be used).Other excipients are known in the art and can be found in Powell et al.,Compendium of Excipients fir Parenteral Formulations (1998), PDA J.Pharm. Sci. Technology, 52:238-311.

Buffers

The stability of a protein drug is usually observed to be maximal in anarrow pH range. This pH range of optimal stability needs to beidentified early during pre-formulation studies. Several approaches suchas accelerated stability studies and calorimetric screening studies havebeen demonstrated to be useful in this endeavor (Remmele R. L. Jr., etal., Biochemistry, 38(16): 5241-7 (1999)). Once a formulation isfinalized, the drug product must be manufactured and maintained within apredefined specification throughout its shelf-life. Hence, bufferingagents are almost always employed to control pH in the formulation.

Organic acids, phosphates and Tris have been employed routinely asbuffers in protein formulations (Table B). The buffer capacity of thebuffering species is maximal at a pH equal to the pKa and decreases aspH increases or decreases away from this value. Ninety percent of thebuffering capacity exists within one pH unit of its pKa. Buffer capacityalso increases proportionally with increasing buffer concentration.

Several factors need to be considered when choosing a buffer. First andforemost, the buffer species and its concentration need to be definedbased on its pKa and the desired formulation pH. Equally important is toensure that the buffer is compatible with the protein drug, otherformulation excipients, and does not catalyze any degradation reactions.Recently, polyanionic carboxylate buffers such as citrate and succinatehave been shown to form covalent adducts with the side chain residues ofproteins. A third important aspect to be considered is the sensation ofstinging and irritation the buffer may induce. For example, citrate isknown to cause stinging upon injection (Laursen T, et al., Basic ClinPharmacol Toxicol., 98(2): 218-21 (2006)). The potential for stingingand irritation is greater for drugs that are administered via the SC orIM routes, where the drug solution remains at the site for a relativelylonger period of time than when administered by the IV route where theformulation gets diluted rapidly into the blood upon administration. Forformulations that are administered by direct IV infusion, the totalamount of buffer (and any other formulation component) needs to bemonitored. One has to be particularly careful about potassium ionsadministered in the form of the potassium phosphate buffer, which caninduce cardiovascular effects in a patient (Hollander-Rodriguez J C, etal., Am. Fam. Physician., 73(2): 283-90 (2006)).

Buffers for lyophilized formulations need additional consideration. Somebuffers like sodium phosphate can crystallize out of the proteinamorphous phase during freezing resulting in rather large shifts in pH.Other common buffers such as acetate and imidazole should be avoidedsince they may sublime or evaporate during the lyophilization process,thereby shifting the pH of formulation during lyophilization or afterreconstitution.

TABLE B Commonly used buffering agents and their pK_(a) values BufferpK_(a) Example drug product Acetate 4.8 Neupogen, Neulasta SuccinatepK_(a1) = 4.8, pK_(a2) = 5.5 Actimmune Citrate pK_(a1) = 3.1, pK_(a2) =4.8, Humira pK_(a3) = 6.4 Histidine 6.0 Xolair (imidazole) phosphatepK_(a1) = 2.15, pK_(a2) = 7.2, Enbrel (liquid formulation) pK_(a3) =12.3 Tris 8.1 Leukine

The buffer system present in the compositions is selected to bephysiologically compatible and to maintain a desired pH in thereconstituted solution as well as in the solution before lyophilization.In one embodiment, the pH of the solution prior to lyophilization isbetween pH 2.0 and pH 12.0. For example, in one embodiment the pH of thesolution prior to lyophilization is 2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5,3.7, 4.0, 4.3, 4.5, 4.7, 5.0, 5.3, 5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0,7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7, 9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5,10.7, 11.0, 11.3, 11.5, 11.7, or 12.0. In another embodiment, the pH ofthe reconstituted solution is between 4.5 and 9.0. In one embodiment,the pH in the reconstituted solution is 4.5, 4.7, 5.0, 5.3, 5.5, 5.7,6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7, or 9.0.

In one embodiment, the pH buffering agent used in the formulation is anamino acid or mixture of amino acids. In one aspect, the pH bufferingagent is histidine or a mixture of amino acids one of which ishistidine.

The pH buffering compound may be present in any amount suitable tomaintain the pH of the formulation at a predetermined level. In oneembodiment, when the pH buffering agent is an amino acid, theconcentration of the amino acid is between 0.1 mM and 1000 mM (1 M). Inone embodiment, the pH buffering agent is at least 0.1, 0.5, 0.7, 0.80.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or900 mM. In another embodiment, the concentration of the pH bufferingagent is between 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM and 100mM. In still another embodiment, the concentration of the pH bufferingagent is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 30, or 40 mM and 50 mM. In yet another embodiment, the concentrationof the pH buffering agent is 10 mM.

Other exemplary pH buffering agents used to buffer the formulation asset out herein include, but are not limited to glycine, histidine,glutamate, succinate, phosphate, acetate, and aspartate.

Stabilizers and Bulking Agents

Bulking agents are typically used in lyophilized formulations to enhanceproduct elegance and to prevent blowout. Conditions in the formulationare generally designed so that the bulking agent crystallizes out of thefrozen amorphous phase (either during freezing or annealing above theTg′) giving the cake structure and bulk. Mannitol and glycine areexamples of commonly used bulking agents.

Stabilizers include a class of compounds that can serve ascryoprotectants, lyoprotectants, and glass forming agents.Cryoprotectants act to stabilize proteins during freezing or in thefrozen state at low temperatures (P. Cameron, ed., Good PharmaceuticalFreeze-Drying Practice, Interpharm Press, Inc., Buffalo Grove, Ill.,(1997)). Lyoprotectants stabilize proteins in the freeze-dried soliddosage form by preserving the native-like conformational properties ofthe protein during dehydration stages of freeze-drying. Glassy stateproperties have been classified as “strong” or “fragile” depending ontheir relaxation properties as a function of temperature. It isimportant that cryoprotectants, lyoprotectants, and glass forming agentsremain in the same phase with the protein in order to impart stability.Sugars, polymers, and polyols fall into this category and can sometimesserve all three roles.

Polyols encompass a class of excipients that includes sugars, (e.g.mannitol, sucrose, sorbitol), and other polyhydric alcohols (e.g.,glycerol and propylene glycol). The polymer polyethylene glycol (PEG) isincluded in this category. Polyols are commonly used as stabilizingexcipients and/or isotonicity agents in both liquid and lyophilizedparenteral protein formulations. With respect to the Hofineister series,the polyols are kosmotropic and are preferentially excluded from theprotein surface. Polyols can protect proteins from both physical andchemical degradation pathways. Preferentially excluded co-solventsincrease the effective surface tension of solvent at the proteininterface whereby the most energetically favorable protein conformationsare those with the smallest surface areas.

Mannitol is a popular bulking agent in lyophilized formulations becauseit crystallizes out of the amorphous protein phase during freeze-dryinglending structural stability to the cake (e.g. Leukine®, Enbrel®—Lyo,Betaseron®). It is generally used in combination with a cryo and/orlyoprotectant like sucrose. Because of the propensity of mannitol tocrystallize under frozen conditions, sorbitol and sucrose are thepreferred tonicity agents/stabilizers in liquid formulations to protectthe product against freeze-thaw stresses encountered during transport orwhen freezing bulk prior to manufacturing. Sorbitol and sucrose are farmore resistant to crystallization and therefore less likely to phaseseparate from the protein. It is interesting to note that while mannitolhas been used in tonicifying amounts in several marketed liquidformulations such as Actimmune®, Forteo®, and Rebif®, the product labelsof these drugs carry a ‘Do Not Freeze’ warning. The use of reducingsugars (containing free aldehyde or ketone groups) such as glucose andlactose should be avoided because they can react and glycate surfacelysine and arginine residues of proteins via the Maillard reaction ofaldehydes and primary amines (Chevalier F, et al., Nahrung, 46(2): 58-63(2002); Humeny A, et al., J Agric Food Chem. 50(7): 2153-60 (2002)).Sucrose can hydrolyze to fructose and glucose under acidic conditions(Kautz C. F. and Robinson A. L., JACS, 50(4) 1022-30 (1928)), andconsequently may cause glycation.

The polymer polyethylene glycol (PEG) could stabilize proteins by twodifferent temperature dependent mechanisms. At lower temperatures, it ispreferentially excluded from the protein surface but has been shown tointeract with the unfolded form of the protein at higher temperaturegiven its amphipathic nature (Lee L. L., and Lee J. C., Biochemistry,26(24): 7813-9 (1987)). Thus at lower temperatures it may protectproteins via the mechanism of preferential exclusion, but at highertemperatures possibly by reducing the number of productive collisionsbetween unfolded molecules. PEG is also a cryoprotectant and has beenemployed in Recombinate®, a lyophilized formulation of recombinantAntihemophilic Factor, which utilizes PEG 3350 at a concentration of 1.5mg/mL. The low molecular weight liquid PEGs (PEG 300-600) can becontaminated with peroxides and cause protein oxidation. If used, theperoxide content in the raw material must be minimized and controlledthroughout its shelf-life. The same holds true for polysorbates.

In a particular embodiment of the present compositions, a stabilizer (ora combination of stabilizers) is added to the lyophilization formulationto prevent or reduce lyophilization-induced or storage-inducedaggregation and chemical degradation. A hazy or turbid solution uponreconstitution indicates that the protein has precipitated. The term“stabilizer” means an excipient capable of preventing aggregation orother physical degradation, as well as chemical degradation (forexample, autolysis, deamidation, oxidation, etc.) in an aqueous andsolid state. Stabilizers that are conventionally employed inpharmaceutical compositions include, but are not limited to, sucrose,trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose,gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol,sorbitol, glycine, arginine HCL, poly-hydroxy compounds, includingpolysaccharides such as dextran, starch, hydroxyethyl starch,cyclodextrins, N-methylpyrollidene, cellulose and hyaluronic acid,sodium chloride, [Carpenter et al., Develop. Biol. Standard 74:225,(1991)]. In one embodiment, the stabilizer is incorporated in aconcentration of about 0% to about 40% w/v. In another embodiment, thestabilizer is incorporated in a concentration of at least 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40%w/v. In another embodiment, the stabilizer is incorporated in aconcentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9% to about 10% w/v. Instill another embodiment, the stabilizer is incorporated in aconcentration of about 2% to about 4% w/v. In yet another embodiment,the stabilizer is incorporated in a concentration of about 2% W/V.

If desired, the lyophilized compositions also include appropriateamounts of bulking and osmolarity regulating agents suitable for forminga lyophilized “cake”. Bulking agents may be either crystalline (forexample, mannitol, glycine) or amorphous (for example, sucrose, polymerssuch as dextran, polyvinylpyrolidone, carboxymethylcellulose). Otherexemplary bulking agents include lactose, sorbitol, trehalose, orxylitol. In one embodiment, the bulking agent is mannitol. In a furtherembodiment, the bulking agent is incorporated in a concentration ofabout 0% to about 10% w/v. In another embodiment, the bulking agent isincorporated in a concentration of at least 0.2, 0.5, 0.7, 1.0, 1.5,2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5,9.0, or 9.5% w/v. In a yet further embodiment in a concentration ofabout 1, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5% to 5.0% w/v, to produce amechanically and pharmaceutically stable and elegant cake. In anotherembodiment, the mannitol concentration is 4% w/v.

Surfactants

Protein molecules have a high propensity to interact with surfacesmaking them susceptible to adsorption and denaturation at air-liquid,vial-liquid, and liquid-liquid (silicone oil) interfaces. Thisdegradation pathway has been observed to be inversely dependent onprotein concentration and result in either the formation of soluble andinsoluble protein aggregates or the loss of protein from solution viaadsorption to surfaces. In addition to container surface adsorption,surface-induced degradation is exacerbated with physical agitation, aswould be experienced during shipping and handling of the product.

Surfactants are commonly used in protein formulations to preventsurface-induced degradation. Surfactants are amphipathic molecules withthe capability of out-competing proteins for interfacial positions.Hydrophobic portions of the surfactant molecules occupy interfacialpositions (e.g., air/liquid), while hydrophilic portions of themolecules remain oriented towards the bulk solvent. At sufficientconcentrations (typically around the detergent's critical micellarconcentration), a surface layer of surfactant molecules serve to preventprotein molecules from adsorbing at the interface. Thereby,surface-induced degradation is minimized. The most commonly usedsurfactants are fatty acid esters of sorbitan polyethoxylates, i.e.polysorbate 20 and polysorbate 80 (e.g., Avonex®, Neupogen®, Neulasta®).The two differ only in the length of the aliphatic chain that impartshydrophobic character to the molecules, C-12 and C-18, respectively.Accordingly, polysorbate-80 is more surface-active and has a lowercritical micellar concentration than polysorbate-20. The surfactantpoloxamer 188 has also been used in several marketed liquid productssuch Gonal-F®, Norditropin®, and Ovidrel®.

Detergents can also affect the thermodynamic conformational stability ofproteins. Here again, the effects of a given excipient will be proteinspecific. For example, polysorbates have been shown to reduce thestability of some proteins and increase the stability of others.Detergent destabilization of proteins can be rationalized in terms ofthe hydrophobic tails of the detergent molecules that can engage inspecific binding with partially or wholly unfolded protein states. Thesetypes of interactions could cause a shift in the conformationalequilibrium towards the more expanded protein states (i.e. increasingthe exposure of hydrophobic portions of the protein molecule incomplement to binding polysorbate). Alternatively, if the protein nativestate exhibits some hydrophobic surfaces, detergent binding to thenative state may stabilize that conformation.

Another aspect of polysorbates is that they are inherently susceptibleto oxidative degradation. Often, as raw materials, they containsufficient quantities of peroxides to cause oxidation of protein residueside-chains, especially methionine. The potential for oxidative damagearising from the addition of stabilizer emphasizes the point that thelowest effective concentrations of excipients should be used informulations. For surfactants, the effective concentration for a givenprotein will depend on the mechanism of stabilization. It has beenpostulated that if the mechanism of surfactant stabilization is relatedto preventing surface-denaturation the effective concentration will bearound the detergent's critical micellar concentration. Conversely, ifthe mechanism of stabilization is associated with specificprotein-detergent interactions, the effective surfactant concentrationwill be related to the protein concentration and the stoichiometry ofthe interaction (Randolph T. W., et al., Pharm Biotechnol., 13:159-75(2002)).

Surfactants may also be added in appropriate amounts to prevent surfacerelated aggregation phenomenon during freezing and drying [Chang, B, J.Pharm. Sci. 85:1325, (1996)]. Exemplary surfactants include anionic,cationic, nonionic, zwitterionic, and amphoteric surfactants includingsurfactants derived from naturally-occurring amino acids. Anionicsurfactants include, but are not limited to, sodium lauryl sulfate,dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate,chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecylsulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate,sodium deoxycholate, and glycodeoxycholic acid sodium salt. Cationicsurfactants include, but are not limited to, benzalkonium chloride orbenzethonium chloride, cetylpyridinium chloride monohydrate, andhexadecyltrimethylammonium bromide. Zwitterionic surfactants include,but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Non-ionicsurfactants include, but are not limited to, digitonin, Triton X-100,Triton X-114, TWEEN-20, and TWEEN-80. In another embodiment, surfactantsinclude lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylenehydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate,polysorbate 40, 60, 65 and 80, soy lecithin and other phospholipids suchas DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methylcellulose and carboxymethyl cellulose. Compositions comprising thesesurfactants, either individually or as a mixture in different ratios,are therefore further provided. In one embodiment, the surfactant isincorporated in a concentration of about 0% to about 5% w/v. In anotherembodiment, the surfactant is incorporated in a concentration of atleast 0.001, 0.002, 0.005, 0.007, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, or 4.5% w/v.In another embodiment, the surfactant is incorporated in a concentrationof about 0.001% to about 0.5% w/v. In still another embodiment, thesurfactant is incorporated in a concentration of about 0.004, 0.005,0.007, 0.01, 0.05, or 0.1% w/v to about 0.2% w/v. In yet anotherembodiment, the surfactant is incorporated in a concentration of about0.01% to about 0.1% w/v.

Salts

Salts are often added to increase the ionic strength of the formulation,which can be important for protein solubility, physical stability, andisotonicity. Salts can affect the physical stability of proteins in avariety of ways. Ions can stabilize the native state of proteins bybinding to charged residues on the protein's surface. Alternatively,they can stabilize the denatured state by binding to the peptide groupsalong the protein backbone (—CONH—). Salts can also stabilize theprotein native conformation by shielding repulsive electrostaticinteractions between residues within a protein molecule. Electrolytes inprotein formulations can also shield attractive electrostaticinteractions between protein molecules that can lead to proteinaggregation and insolubility.

The effect of salt on the stability and solubility of proteins variessignificantly with the characteristics of the ionic species. TheHofineister series originated in the 1880's as a way to rank orderelectrolytes based on their ability to precipitate proteins (Cacace M.G., et al., Quarterly Reviews of Biophysics., 30(3): 241-277 (1997)). Inthis report, the Hofmeister series is used to illustrate a scale ofprotein stabilization effects by ionic and non-ionic co-solutes. InTable C, co-solutes are ordered with respect to their general effects onsolution state proteins, from stabilizing (kosmotropic) to destabilizing(chaotropic). In general, the differences in effects across the anionsare far greater than that observed for the cations, and, for both types,the effects are most apparent at higher concentrations than areacceptable in parenteral formulations. High concentrations ofkosmotropes (e.g., >1 molar ammonium sulfate) are commonly used toprecipitate proteins from solution by a process called ‘salting-out’where the kosmotrope is preferentially excluded from the protein surfacereducing the solubility of the protein in it's native (folded)conformation. Removal or dilution of the salt will return the protein tosolution. The term ‘salting-in’ refers to the use of destabilizing ions(e.g., like guanidine and chloride) that increase the solubility ofproteins by solvating the peptide bonds of the protein backbone.Increasing concentrations of the chaotrope will favor the denatured(unfolded) state conformation of the protein as the solubility of thepeptide chain increases. The relative effectiveness of ions to ‘salt-in’and ‘salt-out’ defines their position in the Hofmeister series.

In order to maintain isotonicity in a parenteral formulation, saltconcentrations are generally limited to less than 150 mM for monovalention combinations. In this concentration range, the mechanism of saltstabilization is probably due to screening of electrostatic repulsiveintramolecular forces or attractive intermolecular forces (Debye-Huckelscreening). Interestingly, chaotropic salts have been shown to be moreeffective at stabilizing the protein structure than similarconcentrations of kosmotropes by this mechanism. The chaotropic anionsare believed to bind more strongly than the kosmotropic ions. Withrespect to covalent protein degradation, differential effects of ionicstrength on this mechanism are expected through Debye-Huckel theory.Accordingly, published reports of protein stabilization by sodiumchloride are accompanied by those where sodium chloride acceleratedcovalent degradation. The mechanisms by which salts affect proteinstability are protein specific and may vary significantly as a functionof solution pH. An example where an excipient can be useful in enablingthe delivery of a protein drug is that of some high concentrationantibody formulations. Recently; salts have been shown to be effectivein reducing the viscosity of such formulations (Liu J., et al., J. PharmSci., 94(9): 1928-40 (2005); Erratum in: J. Pharm Sci., 95(1): 234-5.(2006)).

TABLE C The Hofmeister series of salts

Amino Acids

Amino acids have found versatile use in protein formulations as buffers,bulking agents, stabilizers and antioxidants. Histidine and glutamicacid are employed to buffer protein formulations in the pH range of5.5-6.5 and 4.0-5.5 respectively. The imidazole group of histidine has apKa=6.0 and the carboxyl group of glutamic acid side chain has a pK_(a)of 4.3 which makes them suitable for buffering in their respective pHranges. Acetate, the most commonly used buffer in the acidic pH range of4.0-5.5, sublimates during lyophilization and hence should not be usedin freeze-dried formulations. Glutamic acid is particularly useful insuch cases (e.g., Stemgen®). Histidine is commonly found in marketedprotein formulations (e.g., Xolair®, Herceptin®, Recombinate®). Itprovides a good alternative to citrate, a buffer known to sting uponinjection. Interestingly, histidine has also been reported to have astabilizing effect on ABX-IL8 (an IgG2 antibody) with respect toaggregation when used at high concentrations in both liquid andlyophilized presentations (Chen B, et al., Pharm Res., 20(12): 1952-60(2003)). Histidine (up to 60 mM) was also observed to reduce theviscosity of a high concentration formulation of this antibody. However,in the same study, the authors observed increased aggregation anddiscoloration in histidine containing formulations during freeze-thawstudies of the antibody in stainless steel containers. The authorsattributed this to an effect of iron ions leached from corrosion ofsteel containers. Another note of caution with histidine is that itundergoes photo-oxidation in the presence of metal ions (Tomita M, etal., Biochemistry, 8(12): 5149-60 (1969)). The use of methionine as anantioxidant in formulations appears promising; it has been observed tobe effective against a number of oxidative stresses (Lam X M, et al., JPharm Sci., 86(11): 1250-5 (1997)).

The amino acids glycine, proline, serine and alanine have been shown tostabilize proteins by the mechanism of preferential exclusion. Glycineis also a commonly used bulking agent in lyophilized formulations (e.g.,Neumega®, Genotropin®, Humatrope®). It crystallizes out of the frozenamorphous phase giving the cake structure and bulk. Arginine has beenshown to be an effective agent in inhibiting aggregation and has beenused in both liquid and lyophilized formulations (e.g., Activase®,Avonex®, Enbrel® liquid). Furthermore, the enhanced efficiency ofrefolding of certain proteins in the presence of arginine has beenattributed to its suppression of the competing aggregation reactionduring refolding.

Antioxidants

Oxidation of protein residues arises from a number of different sources.Beyond the addition of specific antioxidants, the prevention ofoxidative protein damage involves the careful control of a number offactors throughout the manufacturing process and storage of the productsuch as atmospheric oxygen, temperature, light exposure, and chemicalcontamination. The most commonly used pharmaceutical antioxidants arereducing agents, oxygen/free-radical scavengers, or chelating agents.Antioxidants in therapeutic protein formulations must be water-solubleand remain active throughout the product shelf-life. Reducing agents andoxygen/free-radical scavengers work by ablating active oxygen species insolution. Chelating agents such as EDTA can be effective by bindingtrace metal contaminants that promote free-radical formation. Forexample, EDTA was utilized in the liquid formulation of acidicfibroblast growth factor to inhibit the metal ion catalyzed oxidation ofcysteine residues. EDTA has been used in marketed products like Kineret®and Ontak®.

In addition to evaluating the effectiveness of various excipients atpreventing protein oxidation, formulation scientists must be aware ofthe potential for the antioxidants themselves to induce other covalentor physical changes to the protein. A number of such cases have beenreported in the literature. Reducing agents (like glutathione) can causedisruption of intramolecular disulfide linkages, which can lead todisulfide shuffling. In the presence of transition metal ions, ascorbicacid and EDTA have been shown to promote methionine oxidation in anumber of proteins and peptides (Akers M J, and Defelippis M R. Peptidesand Proteins as Parenteral Solutions. In: Pharmaceutical FormulationDevelopment of Peptides and Proteins. Sven Frokjaer, Lars Hovgaard,editors. Pharmaceutical Science. Taylor and Francis, U K (1999));Fransson J. R., J. Pharm. Sci. 86(9): 4046-1050 (1997); Yin J, et al.,Pharm Res., 21(12): 2377-83 (2004)). Sodium thiosulfate has beenreported to reduce the levels of light and temperature inducedmethionine-oxidation in rhuMab HER2; however, the formation of athiosulfate-protein adduct was also reported in this study (Lam X M,Yang J Y, et al., J Pharm Sci. 86(11): 1250-5 (1997)). Selection of anappropriate antioxidant is made according to the specific stresses andsensitivities of the protein.

Metal Ions

In general, transition metal ions are undesired in protein formulationsbecause they can catalyze physical and chemical degradation reactions inproteins. However, specific metal ions are included in formulations whenthey are co-factors to proteins and in suspension formulations ofproteins where they form coordination complexes (e.g., zinc suspensionof insulin). Recently, the use of magnesium ions (10-120 mM) has beenproposed to inhibit the isomerization of aspartic acid to isoasparticacid (WO 2004039337).

Two examples where metal ions confer stability or increased activity inproteins are human deoxyribonuclease (rhDNase, Pulmozyme®), and FactorVIII. In the case of rhDNase, Ca⁺² ions (up to 100 mM) increased thestability of the enzyme through a specific binding site (Chen B, et al.,J Pharm Sci., 88(4): 477-82 (1999)). In fact, removal of calcium ionsfrom the solution with EGTA caused an increase in deamidation andaggregation. However, this effect was observed only with Ca⁺² ions;other divalent cations —Mg⁺², Mn⁺² and Zn⁺² were observed to destabilizerhDNase. Similar effects were observed in Factor VIII. Ca⁺² and Sr⁺²ions stabilized the protein while others like Mg⁺², Mn⁺² and Zn⁺², Cu⁺²and Fe⁺2 destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm.,155, 121-131 (1997). In a separate study with Factor VIII, a significantincrease in aggregation rate was observed in the presence of Al⁺³ ions(Derrick T S, et al., J. Pharm. Sci., 93(10): 2549-57 (2004)). Theauthors note that other excipients like buffer salts are oftencontaminated with Al⁺³ ions and illustrate the need to use excipients ofappropriate quality in formulated products.

Preservatives

Preservatives are necessary when developing multi-use parenteralformulations that involve more than one extraction from the samecontainer. Their primary function is to inhibit microbial growth andensure product sterility throughout the shelf-life or term of use of thedrug product. Commonly used preservatives include benzyl alcohol, phenoland m-cresol. Although preservatives have a long history of use, thedevelopment of protein formulations that includes preservatives can bechallenging. Preservatives almost always have a destabilizing effect(aggregation) on proteins, and this has become a major factor inlimiting their use in multi-dose protein formulations (Roy S, et al., JPharm Sci., 94(2): 382-96 (2005)).

To date, most protein drugs have been formulated for single-use only.However, when multi-dose formulations are possible, they have the addedadvantage of enabling patient convenience, and increased marketability.A good example is that of human growth hormone (hGH) where thedevelopment of preserved formulations has led to commercialization ofmore convenient, multi-use injection pen presentations. At least foursuch pen devices containing preserved formulations of hGH are currentlyavailable on the market. Norditropin® (liquid, Novo Nordisk), NutropinAQ® (liquid, Genentech) & Genotropin (lyophilized—dual chambercartridge, Pharmacia & Upjohn) contain phenol while Somatrope® (EliLilly) is formulated with m-cresol.

Several aspects need to be considered during the formulation developmentof preserved dosage forms. The effective preservative concentration inthe drug product must be optimized. This requires testing a givenpreservative in the dosage form with concentration ranges that conferanti-microbial effectiveness without compromising protein stability. Forexample, three preservatives were successfully screened in thedevelopment of a liquid formulation for interleukin-1 receptor (Type I),using differential scanning calorimetry (DSC). The preservatives wererank ordered based on their impact on stability at concentrationscommonly used in marketed products (Remmele R L Jr., et al., Pharm Res.,15(2): 200-8 (1998)).

As might be expected, development of liquid formulations containingpreservatives are more challenging than lyophilized formulations.Freeze-dried products can be lyophilized without the preservative andreconstituted with a preservative containing diluent at the time of use.This shortens the time for which a preservative is in contact with theprotein significantly minimizing the associated stability risks. Withliquid formulations, preservative effectiveness and stability have to bemaintained over the entire product shelf-life (˜18-24 months). Animportant point to note is that preservative effectiveness has to bedemonstrated in the final formulation containing the active drug and allexcipient components.

Some preservatives can cause injection site reactions, which is anotherfactor that needs consideration when choosing a preservative. Inclinical trials that focused on the evaluation of preservatives andbuffers in Norditropin, pain perception was observed to be lower informulations containing phenol and benzyl alcohol as compared to aformulation containing m-cresol (Kappelgaard A. M., Horm Res. 62 Suppl3:98-103 (2004)). Interestingly, among the commonly used preservative,benzyl alcohol possesses anesthetic properties (Minogue S C, and Sun DA., Anesth Analg., 100(3): 683-6 (2005)).

Given the teachings and guidance provided herein, those skilled in theart will know what amount or range of excipient can be included in anyparticular formulation to achieve a biopharmaceutical formulation of theinvention that promotes retention in stability of the biopharmaceutical.For example, the amount and type of a salt to be included in abiopharmaceutical formulation of the invention can be selected based onto the desired osmolality (i.e., isotonic, hypotonic or hypertonic) ofthe final solution as well as the amounts and osmolality of othercomponents to be included in the formulation. Similarly, byexemplification with reference to the type of polyol or sugar includedin a formulation, the amount of such an excipient will depend on itsosmolality.

By way of example, inclusion of about 5% sorbitol can achieveisotonicity while about 9% of a sucrose excipient is needed to achieveisotonicity. Selection of the amount or range of concentrations of oneor more excipients that can be included within a biopharmaceuticalformulation of the invention has been exemplified above by reference tosalts, polyols and sugars. However, those skilled in the art willunderstand that the considerations described herein and furtherexemplified by reference to specific excipients are equally applicableto all types and combinations of excipients including, for example,salts, amino acids, other tonicity agents, surfactants, stabilizers,bulking agents, cryoprotectants, lyoprotectants, anti-oxidants, metalions, chelating agents and/or preservatives.

Further, where a particular excipient is reported in a formulation by,e.g., percent (%) w/v, those skilled in the art will recognize that theequivalent molar concentration of that excipient is also contemplated.

Of course, a person having ordinary skill in the art would recognizethat the concentrations of the aforementioned excipients share aninterdependency within a particular formulation. By way of example, theconcentration of a bulking agent may be lowered where, e.g., there is ahigh protein/peptide concentration or where, e.g., there is a highstabilizing agent concentration. In addition, a person having ordinaryskill in the art would recognize that, in order to maintain theisotonicity of a particular formulation in which there is no bulkingagent, the concentration of a stabilizing agent would be adjustedaccordingly (i.e., a “tonicifying” amount of stabilizer would be used).

The compositions are stable for at least two years at 2° C. to 8° C. inthe lyophilized state. This long-term stability is beneficial forextending the shelf life of the pharmaceutical product.

Methods of Preparation

The present invention further contemplates methods for the preparationof therapeutic protein formulations. In one aspect, methods forpreparing a lyophilized therapeutic peptibody formulation comprising thestep of lyophilizing a therapeutic peptibody composition in a buffercomprising a buffering agent, a bulking agent, a stabilizing agent and asurfactant;

The present methods further comprise one or more of the following steps:adding a stabilizing agent to said mixture prior to lyophilizing, addingat least one agent selected from a bulking agent, an osmolarityregulating agent, and a surfactant to said mixture prior tolyophilization. The bulking agent may be any bulking agent describedherein. In one aspect, the bulking agent is mannitol. In anotherembodiment, the stabilizing agent is sucrose. The surfactant may be anysurfactant described herein. In one embodiment, the surfactant ispolysorbate-20.

The standard reconstitution practice for lyophilized material is to addback a volume of pure water or sterile water for injection (WFI)(typically equivalent to the volume removed during lyophilization),although dilute solutions of antibacterial agents are sometimes used inthe production of pharmaceuticals for parenteral administration [Chen,Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)].Accordingly, methods are provided for preparation of reconstitutedtherapeutic peptibodies comprising the step of adding a diluent to alyophilized therapeutic peptibody composition of the invention.

The lyophilized therapeutic peptibody composition may be reconstitutedas an aqueous solution. A variety of aqueous carriers, e.g., sterilewater for injection, water with preservatives for multi dose use, orwater with appropriate amounts of surfactants (for example,polysorbate-20), 0.4% saline, 0.3% glycine, or aqueous suspensions maycontain the active compound in admixture with excipients suitable forthe manufacture of aqueous suspensions. In various aspects, suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

To administer compositions to human or test animals, in one aspect, thecompositions comprises one or more pharmaceutically acceptable carriers.The phrases “pharmaceutically” or “pharmacologically acceptable” referto molecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

The therapeutic peptibody compositions may be administered orally,topically, transdermally, parenterally, by inhalation spray, vaginally,rectally, or by intracranial injection. The term parenteral as usedherein includes subcutaneous injections, intravenous, intramuscular,intracistemal injection, or infusion techniques. Administration byintravenous, intradermal, intramusclar, intramammary, intraperitoneal,intrathecal, retrobulbar, intrapulmonary injection and or surgicalimplantation at a particular site is contemplated as well. Generally,compositions are essentially free of pyrogens, as well as otherimpurities that could be harmful to the recipient.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage will depend on the type of disease to be treated, as definedabove, the severity and course of the disease, whether drug isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the drug, and thediscretion of the attending physician.

Kits

As an additional aspect, the invention includes kits which comprise oneor more lyophilized compounds or compositions packaged in a manner whichfacilitates their use for administration to subjects. In one embodiment,such a kit includes a compound or composition described herein (e.g., acomposition comprising a therapeutic protein or peptide), packaged in acontainer such as a sealed bottle or vessel, with a label affixed to thecontainer or included in the package that describes use of the compoundor composition in practicing the method. In one embodiment, the kitcontains a first container having a lyophilized therapeutic protein orpeptide composition and a second container having a physiologicallyacceptable reconstitution solution for the lyophilized composition. Inone aspect, the compound or composition is packaged in a unit dosageform. The kit may further include a device suitable for administeringthe composition according to a specific route of administration.Preferably, the kit contains a label that describes use of thetherapeutic protein or peptide composition.

Dosages

The dosage regimen involved in a method for treating a conditiondescribed herein will be determined by the attending physician,considering various factors which modify the action of drugs, e.g. theage, condition, body weight, sex and diet of the patient, the severityof any infection, time of administration and other clinical factors. Invarious aspects, the daily regimen is in the range of 0.1-1000 μg of apreparation per kilogram of body weight (calculating the mass of theprotein alone, without chemical modification) or 0.1-150 μg/kg. In someembodiments of the invention, the dose may exceed 1 mg/kg, 3 mg/kg, or10 mg/kg.

Preparations of the invention may be administered by an initial bolusfollowed by a continuous infusion to maintain therapeutic circulatinglevels of drug product. As another example, the inventive compound maybe administered as a one-time dose. Those of ordinary skill in the artwill readily optimize effective dosages and administration regimens asdetermined by good medical practice and the clinical condition of theindividual patient. The frequency of dosing will depend on thepharmacokinetic parameters of the agents and the route ofadministration. The optimal pharmaceutical formulation will bedetermined by one skilled in the art depending upon the route ofadministration and desired dosage. See for example, Remington'sPharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton,Pa. 18042) pages 1435-1712, the disclosure of which is herebyincorporated by reference. Such formulations may influence the physicalstate, stability, rate of in vivo release, and rate of in vivo clearanceof the administered agents. Depending on the route of administration, asuitable dose may be calculated according to body weight, body surfacearea or organ size. Further refinement of the calculations necessary todetermine the appropriate dosage for treatment involving each of theabove mentioned formulations is routinely made by those of ordinaryskill in the art without undue experimentation, especially in light ofthe dosage information and assays disclosed herein, as well as thepharmacokinetic data observed in the human clinical trials discussedabove. Appropriate dosages may be ascertained through use of establishedassays for determining blood level dosages in conjunction withappropriate dose-response data. The final dosage regimen will bedetermined by the attending physician, considering various factors whichmodify the action of drugs, e.g. the drug's specific activity, theseverity of the damage and the responsiveness of the patient, the age,condition, body weight, sex and diet of the patient, the severity of anyinfection, time of administration and other clinical factors. As studiesare conducted, further information will emerge regarding the appropriatedosage levels and duration of treatment for various diseases andconditions.

Structure of Compounds

In General. In preparations in accordance with the invention, a peptideis attached to a vehicle through the N-terminus of the peptide,C-terminus of the peptide, or both, and the resulting structure may befurther modified with a covalently attached WSP which is attached to thevehicle moiety in the vehicle-peptide product. Thus, the therapeuticpeptibody molecules of this invention may be described by the followingformula I:

[(X¹)_(a)—F¹—(X²)_(b)]-(L¹)_(c)-WSP_(d)  I

wherein:

F¹ is a vehicle;

-   -   X¹ is selected from    -   P¹-(L²)_(e)—    -   P²-(L³)_(f)-P¹-(L²)_(e)—    -   P³-(L⁴)_(g)-P²-(L³)_(f)-P¹-(L²)_(e)- and    -   P⁴-(L¹)_(h)-P³-(L⁴)_(g)-P²-(L³)-P¹-(L²)_(e)-

X² is selected from:

-   -   -(L²)_(e)-P¹,    -   -(L²)_(e)-P¹-(L³)_(f)-P²,    -   -(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³, and    -   -(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³-(L⁵)_(h)-P⁴

wherein P¹, P², P³, and P⁴ are each independent sequences ofpharmacologically active peptides;

L¹, L², L³, L⁴, and L⁵ are each independently linkers;

a, b, c, e, f, g, and h are each independently 0 or 1,

provided that at least one of a and b is 1;

d is 0, 1, or greater than 1; and

WSP is a water soluble polymer, the attachment of which is effected atany reactive moiety in F¹.

Thus, compound I comprises compounds of the formulae

[X¹—F¹]-(L¹)_(c)-WSP_(d)  II

including multimers thereof, wherein F¹ is an Fc domain and is attachedat the C-terminus of X¹, and one or more WSP is attached to the Fcdomain, optionally through linker L¹;

[F¹—X²]-(L¹)_(c)-WSP_(d)  III

including multimers thereof, wherein F¹ is an Fc domain and is attachedat the N-terminus of X², and one or more WSP is attached to the Fcdomain, optionally through linker L¹;

[F¹-(L¹)_(e)-P¹]-(L¹)_(c)-WSP_(d)  IV

including multimers thereof, wherein F¹ is an Fc domain and is attachedat the N-terminus of -(L¹)_(c)-P¹ and one or more WSP is attached to theFc domain, optionally through linker L¹; and

[F¹-(L¹)_(e)-P¹-(L²)_(f)-P²]-(L¹)_(c)-WSP_(d)  V

including multimers thereof, wherein F¹ is an Fc domain and is attachedat the N-terminus of -L¹-P¹-L²-P² and one or more WSP is attached to theFc domain, optionally through linker L¹.

In one embodiment, F¹ is an Fc domain and is attached to either theN-terminus or C-terminus of a peptide. In a related embodiment, the Fcis linked into a dimeric form as described herein to which 2 (or more)peptides are attached. The peptides may be homodimeric (i.e., the sameamino acid sequence), or heterodynammic (i.e., different amino acidsequences that bind the same target or that bind different targets).

In another embodiment, Fc-Loops comprising a peptide(s) are provided.Fc-Loops comprising a peptide(s) are prepared in a process in which atleast one biologically active peptide is incorporated as an internalsequence into an Fc domain. Such an internal sequence may be added byinsertion (i.e., between amino acids in the previously existing Fcdomain) or by replacement of amino acids in the previously existing Fcdomain (i.e., removing amino acids in the previously existing Fc domainand adding peptide amino acids). In the latter case, the number ofpeptide amino acids added need not correspond to the number of aminoacids removed from the previously existing Fc domain. For example, inone aspect, a molecule in which 10 amino acids are removed and 15 aminoacids are added is provided. Pharmacologically active compounds providedare prepared by a process comprising: a) selecting at least one peptidethat modulates the activity of a protein of interest; and b) preparing apharmacologic agent comprising an amino acid sequence of the selectedpeptide as an internal sequence of an Fc domain. This process may beemployed to modify an Fc domain that is already linked through an N- orC-terminus or sidechain to a peptide, e.g., as described in U.S. Pat.App. Nos. 2003/0195156, 2003/0176352, 2003/0229023, and 2003/0236193,and international publication numbers WO 00/24770 and WO 04/026329. Theprocess described in U.S. Patent Application Publication No.US2006/0140934 may also be employed to modify an Fc domain that is partof an antibody. In this way, different molecules can be produced thathave additional functionalities, such as a binding domain to a differentepitope or an additional binding domain to the precursor molecule'sexisting epitope. Molecules comprising an internal peptide sequence arealso referred to as “Fc internal peptibodies” or “Fc internal peptidemolecules.”

The Fc internal peptide molecules may include more than one peptidesequence in tandem in a particular internal region, and they may includefurther peptides in other internal regions. While the putative loopregions are preferred, insertions in any other non-terminal domains ofthe Fc are also considered part of this invention. Variants andderivatives of the above compounds (described below) are alsoencompassed by this invention.

The compounds of this invention may be prepared by standard syntheticmethods, recombinant DNA techniques, or any other methods of preparingpeptides and fusion proteins.

A use contemplated for Fc internal peptide molecules is as a therapeuticor a prophylactic agent. A selected peptide may have activity comparableto—or even greater than—the natural ligand mimicked by the peptide. Inaddition, certain natural ligand-based therapeutic agents might induceantibodies against the patient's own endogenous ligand. In contrast, theunique sequence of the vehicle-linked peptide avoids this pitfall byhaving little or typically no sequence identity with the natural ligand.Furthermore, the Fc internal peptibodies may have advantages inrefolding and purification over N- or C-terminally linked Fc molecules.Further still, Fc internal peptibodies may be more stable in boththermodynamically, due to the stabilization of chimeric domains, andchemically, due to increased resistance to proteolytic degradation fromamino- and carboxy-peptidases. Fc internal peptibodies may also exhibitimproved pharmacokinetic properties.

Peptides. Any number of peptides may be used in conjunction with thepresent invention. Of particular interest are peptides that mimic theactivity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL-1ra, CTLA4,TRAIL, TNF, VEGF, MMP, myostatin, integrin, OPG, OPG-L, NGF, TALL-1,Ang-2 binding partner(s), TGF-α, and TGF-β. Peptide antagonists are alsoof interest, particularly those antagonistic to the activity of TNF, anyof the interleukins (IL-1, 2, 3, . . . ), and proteins involved incomplement activation (e.g., C3b). Targeting peptides are also ofinterest, including tumor-homing peptides, membrane-transportingpeptides, and the like. All of these classes of peptides may bediscovered by methods described in the references cited in thisspecification and other references.

Phage display, in particular, is useful in generating peptides for usein the present invention. It has been stated that affinity selectionfrom libraries of random peptides can be used to identify peptideligands for any site of any gene product. Dedman et al. (1993), J. Biol.Chem. 268: 23025-30. Phage display is particularly well suited foridentifying peptides that bind to such proteins of interest as cellsurface receptors or any proteins having linear epitopes. Wilson et al.(1998), Can. J. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc.Today 3: 370-8. Such proteins are extensively reviewed in Herz et al.(1997), J. Receptor & Signal Transduction Res. 17(5): 671-776, which ishereby incorporated by reference. Such proteins of interest arepreferred for use in this invention.

By way of example and without limitation, a group of peptides that bindto cytokine receptors are provided. Cytokines have recently beenclassified according to their receptor code. See Inglot (1997), ArchivumImmunologiae et Therapiae Experimentalis 45: 353-7, which is herebyincorporated by reference. Among these receptors are the CKRs (family Iin Table 3). The receptor classification appears in Table 3.

TABLE 3 Cytokine Receptors Classified by Receptor Code Cytokines(ligands) Receptor Type family subfamily family subfamily I.Hematopoietic 1. IL-2, IL-4, IL-7, I. Cytokine R 1. shared γCr, IL-cytokines IL-9, IL-13, IL-15 (CKR) 9R, IL-4R 2. IL-3, IL-5, GM- 2.shared GP 140 CSF βR 3. IL-6, IL-11, IL- 3. 3. shared RP 130, 12, LIF,OSM, IL-6 R, Leptin R CNTF, Leptin (OB) 4. “single chain” R, 4. G-CSF,EPO, GCSF-R, TPO-R, TPO, PRL, GH GH-R 5. IL-17, HVS-IL- 5. other R² 17II. IL-10 ligands IL-10, BCRF-1, II. IL-10 R HSV-IL-10 III.Interferons 1. IFN-α1, α2, α4, III. Interferon R 1. IFNAR m, t, IFN-β³2. IFNGR 2. IFN-γ IV. IL-1 and IL-1 1. IL-1α, IL-1β, IL- IV. IL-1R 1.IL-1R, IL- like ligands 1Ra 1RAcP 2. IL-18, IL-18BP 2. IL-18R, IL-18RAcP V. TNF family 1. TNF-α, TNF-β 3. NGF/TNF R⁴ TNF-RI, AGP-3R, (LT),FASL, CD40 DR4, DR5, OX40, L, CD30L, CD27 L, OPG, TACI, CD40, OX40L,OPGL, FAS, ODR TRAIL, APRIL, AGP-3, BLys, TL5, Ntn-2, KAY, Neutrokine-αVI. Chemokines 1. α chemokines: 4. Chemokine R 1. CXCR IL-8, GRO α, β,γ, 2. CCR IF-10, PF-4, SDF-1 3. CR 2. β chemokines: 4. DARC⁵ MIP1α,MIP1β, MCP-1,2,3,4, RANTES, eotaxin 3. γ chemokines: lymphotactin VII.Growth factors 1.1 SCF, M-CSF, VII. RKF 1. TK sub-family PDGF-AA, AB,BB, 1.1 IgTK III R, KDR, FLT-1, FLT- VEGF-RI, VEGF-RII 3L, VEGF, SSV-1.2 IgTK IV R PDGF, HGF, SF 1.3 Cysteine-rich 1.2 FGFα, FGFβ TK-I 1.3EGF, TGF-α, 1.4 Cysteine rich VV-F19 (EGF-like) TK-II, IGF-RI 1.4 IGF-I,IGF-II, 1.5 Cysteine knot Insulin TK V 1.5 NGF, BDNF, 2.Serine-threonine NT-3, NT-4⁶ kinase subfamily 2. TGF-β1,β2,β3 (STKS)⁷¹IL-17R - belongs to CKR family but is unassigned to 4 indicatedsubjamilies. ²Other IFN type I subtypes remain unassigned. Hematopoieticcytokines, IL-10 ligands and interferons do not possess functionalintrinsic protein kinases. The signaling molecules for the cytokines areJAK's, STATs and related non-receptor molecules. IL-14, IL-16 and IL-18have been cloned but according to the receptor code they remainunassigned. ³TNF receptors use multiple, distinct intracellularmolecules for signal transduction including “death domain” of FAS R and55 kDa TNF-□R that participates in their cytotoxic effects. NGF/TNF Rcan bind both NGF and related factors as well as TNF ligands. Chemokinereceptors are seven transmembrane (7TM, serpentine) domain receptors.They are G protein-coupled. ⁴The Duffy blood group antigen (DARC) is anerythrocyte receptor that can bind several different chemokines. IL-1Rbelongs to the immunoglobulin superfamily but their signal transductionevents characteristics remain unclear. ⁵The neurotrophic cytokines canassociate with NGF/TNF receptors also. ⁶STKS may encompass many otherTGF-β-related factors that remain unassigned. The protein kinases areintrinsic part of the intracellular domain of receptor kinase family(RKF). The enzymes participate in the signals transmission via thereceptors.

Other proteins of interest as targets for peptide generation in thepresent invention include the following:

αvβ3

αVβ1

Ang-2

B7

B7RP1

CRP1

Calcitonin

CD28

CETP

cMet

Complement factor B

C4b CTLA4 Glucagon Glucagon Receptor LIPG MPL

splice variants of molecules preferentially expressed on tumor cells;e.g., CD44, CD30 unglycosylated variants of mucin and Lewis Y surfaceglycoproteins CD19, CD20, CD33, CD45prostate specific membrane antigen and prostate specific cell antigenmatrix metalloproteinases (MMPs), both secreted and membrane-bound(e.g., MMP-9)

Cathepsins

TIE-2 receptorheparanaseurokinase plasminogen activator (UPA), UPA receptorparathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP),PTH-RI, PTH-RII

Her2 Her3 Insulin Myostatin TALL-1

Nerve growth factorIntegrins and receptorsSelectins and receptors thereof.Cell adhesion molecules and receptors thereof.

Exemplary peptides appear in Tables 4 through 38 below. These peptidesmay be prepared by any methods disclosed in the art, many of which arediscussed herein. In most tables that follow, single letter amino acidabbreviations are used. The “X” in these sequences (and throughout thisspecification, unless specified otherwise in a particular instance)means that any of the 20 naturally occurring amino acid residues may bepresent. Any of these peptides may be linked in tandem (i.e.,sequentially), with or without linkers, and a few tandem-linked examplesare provided in the table. Linkers are listed as “A” and may be any ofthe linkers described herein. Tandem repeats and linkers are shownseparated by dashes for clarity. Any peptide containing a cysteinylresidue may be cross-linked with another Cys-containing peptide, eitheror both of which may be linked to a vehicle. A few cross-linked examplesare provided in the table. Any peptide having more than one Cys residuemay form an intrapeptide disulfide bond, as well; see, for example,EPO-mimetic peptides in Table 5. A few examples of intrapeptidedisulfide-bonded peptides are specified in the table. Any of thesepeptides may be derivatized as described herein, and a few derivatizedexamples are provided in the table. Derivatized peptides in the tablesare exemplary rather than limiting, as the associated underivatizedpeptides may be employed in this invention, as well. For derivatives inwhich the carboxyl terminus may be capped with an amino group, thecapping amino group is shown as —NH₂. For derivatives in which aminoacid residues are substituted by moieties other than amino acidresidues, the substitutions are denoted by σ, which signifies any of themoieties described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9and Cuthbertson et al. (1997), J. Med. Chem. 40: 2876-82, which areincorporated by reference. The J substituent and the Z substituents (Z₅,Z₆, . . . Z₄₀) are as defined in U.S. Pat. Nos. 5,608,035, 5,786,331,and 5,880,096, which are incorporated by reference. For the EPO-mimeticsequences (Table 5), the substituents X₂ through X₁₁ and the integer “n”are as defined in WO 96/40772, which is incorporated by reference. Alsofor the EPO-mimetic sequences, the substituents X_(na), X_(1a), X_(2a),X_(3a), X_(4a), X_(5a) and X_(ca) follow the definitions of X_(n), X₁,X₂, X₃, X₄, X₅, and X_(c), respectively, of WO 99/47151, which is alsoincorporated by reference. The substituents “Ψ” “Θ” and “+” are asdefined in Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4,which is hereby incorporated by reference. X₄, X₅, X₆, and X₇ are asdefined in U.S. Pat. No. 5,773,569, which is hereby incorporated byreference, except that: for integrin-binding peptides, X₁, X₂, X₃, X₄,X₅, X₆, X₇, and X₈ are as defined in International applications WO95/14714, published Jun. 1, 1995 and WO 97/08203, published Mar. 6,1997, which are also incorporated by reference; and for VIP-mimeticpeptides, X₁, X₁′, X₁″, X₂, X₃, X₄, X₅, X₆ and Z and the integers m andn are as defined in WO 97/40070, published Oct. 30, 1997, which is alsoincorporated by reference. Xaa and Yaa below are as defined in WO98/09985, published Mar. 12, 1998, which is incorporated by reference.AA₁, AA₂, AB₁, AB₂, and AC are as defined in International applicationWO 98/53842, published Dec. 3, 1998, which is incorporated by reference.X¹, X², X³, and X⁴ in Table 17 only are as defined in Europeanapplication EP 0 911 393, published Apr. 28, 1999. Residues appearing inboldface are D-amino acids. All peptides are linked through peptidebonds unless otherwise noted. Abbreviations are listed at the end ofthis specification. In the “SEQ ID NO.” column, “NR” means that nosequence listing is required for the given sequence.

TABLE 4 IL-1 antagonist peptide sequences SEQ ID Sequence/structure NO:Z₁₁Z₇Z₈QZ₅YZ₆Z₉Z₁₀ 3 XXQZ₅YZ₆XX 4 Z₇XQZ₅YZ₆XX 5 Z₇Z₈QZ₅YZ₆Z₉Z₁₀ 6Z₁₁Z₇Z₈QZ₅YZ₆Z₉Z₁₀ 7 Z₁₂Z₁₃Z₁₄Z₁₅Z₁₆Z₁₇Z₁₈Z₁₉Z₂₀Z₂₁Z₂₂Z₁₁Z₇Z₈QZ₅YZ₆ 8Z₉Z₁₀L Z₂₃NZ₂₄Z₃₉Z₂₅Z₂₆Z₂₇Z₂₈Z₂₉Z₃₀Z₄₀ 9 TANVSSFEWTPYYWQPYALPL 10SWTDYGYWQPYALPISGL 11 ETPFTWEESNAYYWQPYALPL 12 ENTYSPNWADSMYWQPYALPL 13SVGEDHNFWTSEYWQPYALPL 14 DGYDRWRQSGERYWQPYALPL 15 FEWTPGYWQPY 16FEWTPGYWQHY 17 FEWTPGWYQJY 18 AcFEWTPGWYQJY 19 FEWTPGWpYQJY 20FAWTPGYWQJY 21 FEWAPGYWQJY 22 FEWVPGYWQJY 23 FEWTPGYWQJY 24AcFEWTPGYWQJY 25 FEWTPaWYQJY 26 FEWTPSarWYQJY 27 FEWTPGYYQPY 28FEWTPGWWQPY 29 FEWTPNYWQPY 30 FEWTPvYWQJY 31 FEWTPecGYWQJY 32FEWTPAibYWQJY 33 FEWTSarGYWQJY 34 FEWTPGYWQPY 35 FEWTPGYWQHY 36FEWTPGWYQJY 37 AcFEWTPGWYQJY 38 FEWTPGW-pY-QJY 39 FAWTPGYWQJY 40FEWAPGYWQJY 41 FEWVPGYWQJY 42 FEWTPGYWQJY 43 AcFEWTPGYWQJY 44FEWTPAWYQJY 45 FEWTPSarWYQJY 46 FEWTPGYYQPY 47 FEWTPGWWQPY 48FEWTPNYWQPY 49 FEWTPVYWQJY 50 FEWTPecGYWQJY 51 FEWTPAibYWQJY 52FEWTSarGYWQJY 53 FEWTPGYWQPYALPL 54 1NapEWTPGYYQJY 55 YEWTPGYYQJY 56FEWVPGYYQJY 57 FEWTPSYYQJY 58 FEWTPNYYQJY 59 TKPR 60 RKSSK 61 RKQDK 62NRKQDK 63 RKQDKR 64 ENRKQDKRF 65 VTKFYF 66 VTKFY 67 VTDFY 68 SHLYWQPYSVQ69 TLVYWQPYSLQT 70 RGDYWQPYSVQS 71 VHVYWQPYSVQT 72 RLVYWQPYSVQT 73SRVWFQPYSLQS 74 NMVYWQPYSIQT 75 SVVFWQPYSVQT 76 TFVYWQPYALPL 77TLVYWQPYSIQR 78 RLVYWQPYSVQR 79 SPVFWQPYSIQI 80 WIEWWQPYSVQS 81SLIYWQPYSLQM 82 TRLYWQPYSVQR 83 RCDYWQPYSVQT 84 MRVFWQPYSVQN 85KIVYWQPYSVQT 86 RHLYWQPYSVQR 87 ALVWWQPYSEQI 88 SRVWFQPYSLQS 89WEQPYALPLE 90 QLVWWQPYSVQR 91 DLRYWQPYSVQV 92 ELVWWQPYSLQL 93DLVWWQPYSVQW 94 NGNYWQPYSFQV 95 ELVYWQPYSIQR 96 ELMYWQPYSVQE 97NLLYWQPYSMQD 98 GYEWYQPYSVQR 99 SRVWYQPYSVQR 100 LSEQYQPYSVQR 101GGGWWQPYSVQR 102 VGRWYQPYSVQR 103 VHVYWQPYSVQR 104 QARWYQPYSVQR 105VHVYWQPYSVQT 106 RSVYWQPYSVQR 107 TRVWFQPYSVQR 108 GRIWFQPYSVQR 109GRVWFQPYSVQR 110 ARTWYQPYSVQR 111 ARVWWQPYSVQM 112 RLMFYQPYSVQR 113ESMWYQPYSVQR 114 HFGWWQPYSVHM 115 ARFWWQPYSVQR 116 RLVYWQ PYAPIY 117RLVYWQ PYSYQT 118 RLVYWQ PYSLPI 119 RLVYWQ PYSVQA 120 SRVWYQ PYAKGL 121SRVWYQ PYAQGL 122 SRVWYQ PYAMPL 123 SRVWYQ PYSVQA 124 SRVWYQ PYSLGL 125SRVWYQ PYAREL 126 SRVWYQ PYSRQP 127 SRVWYQ PYFVQP 128 EYEWYQ PYALPL 129IPEYWQ PYALPL 130 SRIWWQ PYALPL 131 DPLFWQ PYALPL 132 SRQWVQ PYALPL 133IRSWWQ PYALPL 134 RGYWQ PYALPL 135 RLLWVQ PYALPL 136 EYRWFQ PYALPL 137DAYWVQ PYALPL 138 WSGYFQ PYALPL 139 NIEFWQ PYALPL 140 TRDWVQ PYALPL 141DSSWYQ PYALPL 142 IGNWYQ PYALPL 143 NLRWDQ PYALPL 144 LPEFWQ PYALPL 145DSYWWQ PYALPL 146 RSQYYQ PYALPL 147 ARFWLQ PYALPL 148 NSYFWQ PYALPL 149RFMYWQPYSVQR 150 AHLFWQPYSVQR 151 WWQPYALPL 152 YYQPYALPL 153 YFQPYALGL154 YWYQPYALPL 155 RWWQPYATPL 156 GWYQPYALGF 157 YWYQPYALGL 158IWYQPYAMPL 159 SNMQPYQRLS 160 TFVYWQPY AVGLPAAETACN 161 TFVYWQPYSVQMTITGKVTM 162 TFVYWQPY SSHXXVPXGFPL 163 TFVYWQPY YGNPQWAIHVRH 164TFVYWQPY VLLELPEGAVRA 165 TFVYWQPY VDYVWPIPIAQV 166 GWYQPYVDGWR 167RWEQPYVKDGWS 168 EWYQPYALGWAR 169 GWWQPYARGL 170 LFEQPYAKALGL 171GWEQPYARGLAG 172 AWVQPYATPLDE 173 MWYQPYSSQPAE 174 GWTQPYSQQGEV 175DWFQPYSIQSDE 176 PWIQPYARGFG 177 RPLYWQPYSVQV 178 TLIYWQPYSVQI 179RFDYWQPYSDQT 180 WHQFVQPYALPL 181 EWDS VYWQPYSVQ TLLR 182 WEQN VYWQPYSVQSFAD 183 SDV VYWQPYSVQ SLEM 184 YYDG VYWQPYSVQ VMPA 185 SDIWYQ PYALPL186 QRIWWQ PYALPL 187 SRIWWQ PYALPL 188 RSLYWQ PYALPL 189 TIIWEQ PYALPL190 WETWYQ PYALPL 191 SYDWEQ PYALPL 192 SRIWCQ PYALPL 193 EIMFWQ PYALPL194 DYVWQQ PYALPL 195 MDLLVQ WYQPYALPL 196 GSKVIL WYQPYALPL 197 RQGANIWYQPYALPL 198 GGGDEP WYQPYALPL 199 SQLERT WYQPYALPL 200 ETWVRE WYQPYALPL201 KKGSTQ WYQPYALPL 202 LQARMN WYQPYALPL 203 EPRSQK WYQPYALPL 204VKQKWR WYQPYALPL 205 LRRHDV WYQPYALPL 206 RSTASI WYQPYALPL 207 ESKEDQWYQPYALPL 208 EGLTMK WYQPYALPL 209 EGSREG WYQPYALPL 210 VIEWWQ PYALPL211 VWYWEQ PYALPL 212 ASEWWQ PYALPL 213 FYEWWQ PYALPL 214 EGWWVQ PYALPL215 WGEWLQ PYALPL 216 DYVWEQ PYALPL 217 AHTWWQ PYALPL 218 FIEWFQ PYALPL219 WLAWEQ PYALPL 220 VMEWWQ PYALPL 221 ERMWQ PYALPL 222 NXXWXX PYALPL223 WGNWYQ PYALPL 224 TLYWEQ PYALPL 225 VWRWEQ PYALPL 226 LLWTQ PYALPL227 SRIWXX PYALPL 228 SDIWYQ PYALPL 229 WGYYXX PYALPL 230 TSGWYQ PYALPL231 VHPYXX PYALPL 232 EHSYFQ PYALPL 233 XXIWYQ PYALPL 234 AQLHSQ PYALPL235 WANWFQ PYALPL 236 SRLYSQ PYALPL 237 GVTFSQ PYALPL 238 SIVWSQ PYALPL239 SRDLVQ PYALPL 240 HWGH VYWQPYSVQ DDLG 241 SWHS VYWQPYSVQ SVPE 242WRDS VYWQPYSVQ PESA 243 TWDA VYWQPYSVQ KWLD 244 TPPW VYWQPYSVQ SLDP 245YWSS VYWQPYSVQ SVHS 246 YWY QPY ALGL 247 YWY QPY ALPL 248 EWI QPY ATGL249 NWE QPY AKPL 250 AFY QPY ALPL 251 FLY QPY ALPL 252 VCK QPY LEWC 253ETPFTWEESNAYYWQPYALPL 254 QGWLTWQDSVDMYWQPYALPL 255FSEAGYTWPENTYWQPYALPL 256 TESPGGLDWAKIYWQPYALPL 257DGYDRWRQSGERYWQPYALPL 258 TANVSSFEWTPGYWQPYALPL 259 SVGEDHNFWTSEYWQPYALPL 260 MNDQTSEVSTFP YWQPYALPL 261 SWSEAFEQPRNL YWQPYALPL 262QYAEPSALNDWG YWQPYALPL 263 NGDWATADWSNY YWQPYALPL 264 THDEHI YWQPYALPL265 MLEKTYTTWTPG YWQPYALPL 266 WSDPLTRDADL YWQPYALPL 267 SDAFTTQDSQAMYWQPYALPL 268 GDDAAWRTDSLT YWQPYALPL 269 AIIRQLYRWSEM YWQPYALPL 270ENTYSPNWADSM YWQPYALPL 271 MNDQTSEVSTFP YWQPYALPL 272 SVGEDHNFWTSEYWQPYALPL 273 QTPFTWEESNAY YWQPYALPL 274 ENPFTWQESNAY YWQPYALPL 275VTPFTWEDSNVF YWQPYALPL 276 QIPFTWEQSNAY YWQPYALPL 277 QAPLTWQESAAYYWQPYALPL 278 EPTFTWEESKAT YWQPYALPL 279 TTTLTWEESNAY YWQPYALPL 280ESPLTWEESSAL YWQPYALPL 281 ETPLTWEESNAY YWQPYALPL 282 EATFTWAESNAYYWQPYALPL 283 EALFTWKESTAY YWQPYALPL 284 STP-TWEESNAY YWQPYALPL 285ETPFTWEESNAY YWQPYALPL 286 KAPFTWEESQAY YWQPYALPL 287 STSFTWEESNAYYWQPYALPL 288 DSTFTWEESNAY YWQPYALPL 289 YIPFTWEESNAY YWQPYALPL 290QTAFTWEESNAY YWQPYALPL 291 ETLFTWEESNAT YWQPYALPL 292 VSSFTWEESNAYYWQPYALPL 293 QPYALPL 294 Py-1-NapPYQJYALPL 295 TANVSSFEWTPG YWQPYALPL296 FEWTPGYWQPYALPL 297 FEWTPGYWQJYALPL 298 FEWTPGYYQJYALPL 299ETPFTWEESNAYYWQPYALPL 300 FTWEESNAYYWQJYALPL 301 ADVL YWQPYA PVTLWV 302GDVAE YWQPYA LPLTSL 303 SWTDYG YWQPYA LPISGL 304 FEWTPGYWQPYALPL 305FEWTPGYWQJYALPL 306 FEWTPGWYQPYALPL 307 FEWTPGWYQJYALPL 308FEWTPGYYQPYALPL 309 FEWTPGYYQJYALPL 310 TANVSSFEWTPGYWQPYALPL 311SWTDYGYWQPYALPISGL 312 ETPFTWEESNAYYWQPYALPL 313 ENTYSPNWADSMYWQPYALPL314 SVGEDHNFWTSEYWQPYALPL 315 DGYDRWRQSGERYWQPYALPL 316 FEWTPGYWQPYALPL317 FEWTPGYWQPY 318 FEWTPGYWQJY 319 EWTPGYWQPY 320 FEWTPGWYQJY 321AEWTPGYWQJY 322 FAWTPGYWQJY 323 FEATPGYWQJY 324 FEWAPGYWQJY 325FEWTAGYWQJY 326 FEWTPAYWQJY 327 FEWTPGAWQJY 328 FEWTPGYAQJY 329FEWTPGYWQJA 330 FEWTGGYWQJY 331 FEWTPGYWQJY 332 FEWTJGYWQJY 333FEWTPecGYWQJY 334 FEWTPAibYWQJY 335 FEWTPSarWYQJY 336 FEWTSarGYWQJY 337FEWTPNYWQJY 338 FEWTPVYWQJY 339 FEWTVPYWQJY 340 AcFEWTPGWYQJY 341AcFEWTPGYWQJY 342 lNap-EWTPGYYQJY 343 YEWTPGYYQJY 344 FEWVPGYYQJY 345FEWTPGYYQJY 346 FEWTPsYYQJY 347 FEWTPnYYQJY 348 SHLY-Nap-QPYSVQM 349TLVY-Nap-QPYSLQT 350 RGDY-Nap-QPYSVQS 351 NMVY-Nap-QPYSIQT 352 VYWQPYSVQ353 VY-Nap-QPYSVQ 354 TFVYWQJYALPL 355 FEWTPGYYQJ-Bpa 356XaaFEWTPGYYQJ-Bpa 357 FEWTPGY-Bpa-QJY 358 AcFEWTPGY-Bpa-QJY 359FEWTPG-Bpa-YQJY 360 AcFEWTPG-Bpa-YQJY 361 AcFE-Bpa-TPGYYQJY 362AcFE-Bpa-TPGYYQJY 363 Bpa-EWTPGYYQJY 364 AcBpa-EWTPGYYQJY 365 VYWQPYSVQ366 RLVYWQPYSVQR 367 RLVY-Nap-QPYSVQR 368 RLDYWQPYSVQR 369 RLVWFQPYSVQR370 RLVYWQPYSIQR 371 DNSSWYDSFLL 372 DNTAWYESFLA 373 DNTAWYENFLL 374PARE DNTAWYDSFLI WC 375 TSEY DNTTWYEKFLA SQ 376 SQIP DNTAWYQSFLL HG 377SPFI DNTAWYENFLL TY 378 EQIY DNTAWYDHFLL SY 379 TPFI DNTAWYENFLL TY 380TYTY DNTAWYERFLM SY 381 TMTQ DNTAWYENFLL SY 382 TI DNTAWYANLVQ TYPQ 383TI DNTAWYERFLA QYPD 384 HI DNTAWYENFLL TYTP 385 SQ DNTAWYENFLL SYKA 386QI DNTAWYERFLL QYNA 387 NQ DNTAWYESFLL QYNT 388 TI DNTAWYENFLL NHNL 389HY DNTAWYERFLQ QGWH 390 ETPFTWEESNAYYWQPYALPL 391 YIPFTWEESNAYYWQPYALPL392 DGYDRWRQSGERYWQPYALPL 393 pY-lNap-pY-QJYALPL 394TANVSSFEWTPGYWQPYALPL 395 FEWTPGYWQJYALPL 396 FEWTPGYWQPYALPLSD 397FEWTPGYYQJYALPL 398 FEWTPGYWQJY 399 AcFEWTPGYWQJY 400 AcFEWTPGWYQJY 401AcFEWTPGYYQJY 402 AcFEWTPaYWQJY 403 AcFEWTPaWYQJY 404 AcFEWTPaYYQJY 405FEWTPGYYQJYALPL 406 FEWTPGYWQJYALPL 407 FEWTPGWYQJYALPL 408TANVSSFEWTPGYWQPYALPL 409 AcFEWTPGYWQJY 410 AcFEWTPGWYQJY 411AcFEWTPGYYQJY 412 AcFEWTPAYWQJY 413 AcFEWTPAWYQJY 414 AcFEWTPAYYQJY 415

TABLE 5 EPO-mimetic peptide sequences SEQ Sequence/structure ID NO:YXCXXGPXTWXCXP 416 YXCXXGPXTWXCXP-YXCXXGPXTWXCXP 417YXCXXGPXTWXCXP-Λ-YXCXXGPXTWXCXP 418

419 GGTYSCHFGPLTWVCKPQGG 420 GGDYHCRMGPLTWVCKPLGG 421GGVYACRMGPITWVCSPLGG 422 VGNYMCHFGPITWVGRPGGG 423 GGLYLCRFGPVTWDCGYKGG424 GGTYSCHFGPLTWVCKPQGG- 425 GGTYSCHFGPLTWVCKPQGGGGTYSCHFGPLTWVCKPQGG-Λ- 426 GGTYSCHFGPLTWVCKPQGG GGTYSCHFGPLTWVCKPQGGSSK427 GGTYSCHFGPLTWVCKPQGGSSK- 428 GGTYSCHFGPLTWVCKPQGGSSKGGTYSCHFGPLTWVCKPQGGSSK-Λ- 429 GGTYSCHFGPLTWVCKPQGGSSK

430 GGTYSCHFGPLTWVCKPQGGSSK(Λ-biotin) 431 CX₄X₅GPX₆TWX₇C 432GGTYSCHGPLTWVCKPQGG 433 VGNYMAHMGPITWVCRPGG 434 GGPHHVYACRMGPLTWIC 435GGTYSCHFGPLTWVCKPQ 436 GGLYACHMGPMTWVCQPLRG 437 TIAQYICYMGPETWECRPSPKA438 YSCHFGPLTWVCK 439 YCHFGPLTWVC 440 X₃X₄X₅GPX₆TWX₇X₈ 441YX₂X₃X₄X₅GPX₆TWX₇X₈ 442 X₁YX₂X₃X₄X₅GPX₆TWX₇X₈X₉X₁₀X₁₁ 443X₁YX₂CX₄X₅GPX₆TWX₇CX₉X₁₀X₁₁ 444 GGLYLCRFGPVTWDCGYKGG 445GGTYSCHFGPLTWVCKLPQGG 446 GGDYHCRMGPLTWVCKPLGG 447 VGNYMCHFGPITWVCRPGGG448 GGVYACRMGPITWVCSPLGG 449 VGNYMAHMGPITWVCRPGG 450 GGTYSCHFGPLTWVCKPQ451 GGLYACHMGPMTWVCQPLRG 452 TIAQYICYMGPETWECRPSPKA 453 YSCHFGPLTWVCK454 YCHFGPLTWVC 455 SCHFGPLTWVCK 456 (AX₂)_(n)X₃X₄X₅GPX₆TWX₇X₈ 457X_(n)CX₁X₂GWVGX₃CX₄X₅WX_(C) 458

TABLE 6 TPO-mimetic peptide sequences SEQ Sequence/structure ID NO:IEGPTLRQWLAARA 459 IEGPTLRQWLAAKA 460 IEGPTLREWLAARA 461IEGPTLRQWLAARA-Λ-IEGPTLRQWLAARA 462 IEGPTLRQWLAAKA-Λ-IEGPTLRQWLAAKA 463

464 IEGPTLRQWLAARA-Λ-K(BrAc)-Λ-IEGPTLRQWLAARA 465IEGPTLRQWLAARA-Λ-K(PEG)-Λ-IEGPTLRQWLAARA 466

467

468 VRDQIXXXL 469 TLREWL 470 GRVRDQVAGW 471 GRVKDQIAQL 472 GVRDQVSWAL473 ESVREQVMKY 474 SVRSQISASL 475 GVRETVYRIHM 476 GVREVIVMHML 477GRVRDQIWAAL 478 AGVRDQILIWL 479 GRVRDQIMLSL 480 GRVRDQI(X)₃L 481CTLRQWLQGC 482 CTLQEFLEGC 483 CTRTEWLHGC 484 CTLREWLHGGFC 485CTLREWVFAGLC 486 CTLRQWLILLGMC 487 CTLAEFLASGVEQC 488 CSLQEFLSHGGYVC 489CTLREFLDPTTAVC 490 CTLKEWLVSHEVWC 491 CTLREWL(X)₂₋₆C 492 REGPTLRQWM 493EGPTLRQWLA 494 ERGPFWAKAC 495 REGPRCVMWM 496 CGTEGPTLSTWLDC 497CEQDGPTLLEWLKC 498 CELVGPSLMSWLTC 499 CLTGPFVTQWLYEC 500 CRAGPTLLEWLTLC501 CADGPTLREWISFC 502 C(X)₁₋₂EGPTLREWL(X)₁₋₂C 503 GGCTLREWLHGGFCGG 504GGCADGPTLREWISFCGG 505 GNADGPTLRQWLEGRRPKN 506 LAIEGPTLRQWLHGNGRDT 507HGRVGPTLREWKTQVATKK 508 TIKGPTLRQWLKSREHTS 509 ISDGPTLKEWLSVTRGAS 510SIEGPTLREWLTSRTPHS 511

TABLE 7 G-CSF-mimetic peptide sequences SEQ Sequence/structure ID NO:EEDCK 512

513 EEDσK 514

515 pGluEDσK 516

517 PicSDσK 518

519 EEDCK-Λ-EEDCK 520 EEDXK-Λ-EEDXK 521

TABLE 8 TNF-antagonist peptide sequences SEQ Sequence/structure ID NO:YCFTASENHCY 522 YCFTNSENHCY 523 YCFTRSENHCY 524 FCASENHCY 525 YCASENHCY526 FCNSENHCY 527 FCNSENRCY 528 FCNSVENRCY 529 YCSQSVSNDCF 530 FCVSNDRCY531 YCRKELGQVCY 532 YCKEPGQCY 533 YCRKEMGCY 534 FCRKEMGCY 535 YCWSQNLCY536 YCELSQYLCY 537 YCWSQNYCY 538 YCWSQYLCY 539 DFLPHYKNTSLGHRP 540

NR

TABLE 9 Integrin-binding peptide sequences SEQ ID Sequence/structure NO:RX₁ETX₂WX₃ 541 RX₁ETX₂WX₃ 542 RGDGX 543 CRGDGXC 544 CX₁X₂RLDX₃X₄C 545CARRLDAPC 546 CPSRLDSPC 547 X₁X₂X₃RGDX₄X₅X₆ 548 CX₂CRGDCX₅C 549CDCRGDCFC 550 CDCRGDCLC 551 CLCRGDCIC 552 X₁X₂DDX₄X₅X₇X₈ 553X₁X₂X₃DDX₄X₅X₆X₇X₈ 554 CWDDGWLC 555 CWDDLWWLC 556 CWDDGLMC 557 CWDDGWMC558 CSWDDGWLC 559 CPDDLWWLC 560 NGR NR GSL NR RGD NR CGRECPRLCQSSC 561CNGRCVSGCAGRC 562 CLSGSLSC 563 RGD NR NGR NR GSL NR NGRAHA 564 CNGRC 565CDCRGDCFC 566 CGSLVRC 567 DLXXL 568 RTDLDSLRTYTL 569 RTDLDSLRTY 570RTDLDSLRT 571 RTDLDSLR 572 GDLDLLKLRLTL 573 GDLHSLRQLLSR 574RDDLHMLRLQLW 575 SSDLHALKKRYG 576 RGDLKQLSELTW 577 RGDLAALSAPPV 578

TABLE 10 Selectin antagonist peptide sequences SEQ ID Sequence/structureNO: DITWDQLWDLMK 579 DITWDELWKIMN 580 DYTWFELWDMMQ 581 QITWAQLWNMMK 582DMTWHDLWTLMS 583 DYSWHDLWEMMS 584 EITWDQLWEVMN 585 HVSWEQLWDIMN 586HITWDQLWRIMT 587 RNMSWLELWEHMK 588 AEWTWDQLWHVMNPAESQ 589 HRAEWLALWEQMSP590 KKEDWLALWRIMSV 591 ITWDQLWDLMK 592 DITWDQLWDLMK 593 DITWDQLWDLMK 594DITWDQLWDLMK 595 CQNRYTDLVAIQNKNE 596 AENWADNEPNNKRNNED 597RKNNKTWTWVGTKKALTNE 598 KKALTNEAENWAD 599 CQXRYTDLVAIQNKXE 600RKXNXXWTWVGTXKXLTEE 601 AENWADGEPNNKXNXED 602 CXXXYTXLVAIQNKXE 603RKXXXXWXWVGTXKXLTXE 604 AXNWXXXEPNNXXXED 605 XKXKTXEAXNWXX 606

TABLE 11 Antipathogenic peptide sequences SEQ ID Sequence/structure NO:GFFALIPKIISSPLFKTLLSAVGSALSSSGGQQ 607 GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE608 GFFALIPKIISSPLFKTLLSAV 609 GFFALIPKIISSPLFKTLLSAV 610KGFFALIPKIISSPLFKTLLSAV 611 KKGFFALIPKIISSPLFKTLLSAV 612KKGFFALIPKIISSPLFKTLLSAV 613 GFFALIPKIIS 614 GIGAVLKVLTTGLPALISWIKRKRQQ615 GIGAVLKVLTTGLPALISWIKRKRQQ 616 GIGAVLKVLTTGLPALISWIKRKRQQ 617GIGAVLKVLTTGLPALISWIKR 618 AVLKVLTTGLPALISWIKR 619 KLLLLLKLLLLK 620KLLLKLLLKLLK 621 KLLLKLKLKLLK 622 KKLLKLKLKLKK 623 KLLLKLLLKLLK 624KLLLKLKLKLLK 625 KLLLLK 626 KLLLKLLK 627 KLLLKLKLKLLK 628 KLLLKLKLKLLK629 KLLLKLKLKLLK 630 KAAAKAAAKAAK 631 KVVVKVVVKVVK 632 KVVVKVKVKVVK 633KVVVKVKVKVK 634 KVVVKVKVKVVK 635 KLILKL 636 KVLHLL 637 LKLRLL 638 KPLHLL639 KLILKLVR 640 KVFHLLHL 641 HKFRILKL 642 KPFHILHL 643 KIIIKIKIKIIK 644KIIIKIKIKIIK 645 KIIIKIKIKIIK 646 KIPIKIKIKIPK 647 KIPIKIKIKIVK 648RIIIRIRIRIIR 649 RIIIRIRIRIIR 650 RIIIRIRIRIIR 651 RIVIRIRIRLIR 652RIIVRIRLRIIR 653 RIGIRLRVRIIR 654 KIVIRIRIRLIR 655 RIAVKWRLRFIK 656KIGWKLRVRIIR 657 KKIGWLIIRVRR 658 RIVIRIRIRLIRIR 659 RIIVRIRLRIIRVR 660RIGIRLRVRIIRRV 661 KIVIRIRARLIRIRIR 662 RIIVKIRLRIIKKIRL 663KIGIKARVRIIRVKII 664 RIIVHIRLRIIHHIRL 665 HIGIKAHVRIIRVHII 666RIYVKIHLRYIKKIRL 667 KIGHKARVHIIRYKII 668 RIYVKPHPRYIKKIRL 669KPGHKARPHIIRYKII 670 KIVIRIRIRLIRIRIRKIV 671 RIIVKIRLRIIKKIRLIKK 672KIGWKLRVRIIRVKIGRLR 673 KIVIRIRIRLIRIRIRKIVKVKRIR 674RFAVKIRLRIIKKIRLIKKIRKRVIK 675 KAGWKLRVRIIRVKIGRLRKIGWKKRVRIK 676RIYVKPHPRYIKKIRL 677 KPGHKARPHIIRYKII 678 KIVIRIRIRLIRIRIRKIV 679RIIVKIRLRIIKKIRLIKK 680 RIYVSKISIYIKKIRL 681 KIVIFTRIRLTSIRIRSIV 682KPIHKARPTIIRYKMI 683 cyclicCKGFFALIPKIISSPLFKTLLSAVC 684CKKGFFALIPKIISSPLFKTLLSAVC 685 CKKKGFFALIPKIISSPLFKTLLSAVC 686CyclicCRIVIRIRIRLIRIRC 687 CyclicCKPGHKARPHIIRYKIIC 688CyclicCRFAVKIRLRIIKKIRLIKKIRKRVIKC 689 KLLLKLLL KLLKC 690 KLLLKLLLKLLK691 KLLLKLKLKLLKC 692 KLLLKLLLKLLK 693

TABLE 12 VIP-mimetic peptide sequences SEQ Sequence/structure ID NO:HSDAVFYDNYTR LRKQMAVKKYLN SILN 694 Nle HSDAVFYDNYTR LRKQMAVKKYLN SILN695 X₁X₁′X₁″X₂ 696 X₃SX₄LN 697

698 KKYL 699 NSILN 700 KKYL 701 KKYA 702 AVKKYL 703 NSILN 704 KKYV 705SILauN 706 KKYLNle 707 NSYLN 708 NSIYN 709 KKYLPPNSILN 710 LauKKYL 711CapKKYL 712 KYL 713 KKYNle 714 VKKYL 715 LNSILN 716 YLNSILN 717 KKYLN718 KKYLNS 719 KKYLNSI 720 KKYLNSIL 721 KKYL 722 KKYDA 723 AVKKYL 724NSILN 725 KKYV 726 SILauN 727 NSYLN 728 NSIYN 729 KKYLNle 730KKYLPPNSILN 731 KKYL 732 KKYDA 733 AVKKYL 734 NSILN 735 KKYV 736 SILauN737 LauKKYL 738 CapKKYL 739 KYL 740 KYL 741 KKYNle 742 VKKYL 743 LNSILN744 YLNSILN 745 KKYLNle 746 KKYLN 747 KKYLNS 748 KKYLNSI 749 KKYLNSIL750 KKKYLD 751 cyc1icCKKYLC 752

753 KKYA 754 WWTDTGLW 755 WWTDDGLW 756 WWDTRGLWVWTI 757 FWGNDGIWLESG 758DWDQFGLWRGAA 759 RWDDNGLWVVVL 760 SGMWSHYGIWMG 761 GGRWDQAGLWVA 762KLWSEQGIWMGE 763 CWSMIIGLWLC 764 GCWDNTGIWVPC 765 DWDTRGLWVY 766SLWDENGAWI 767 KWDDRGLWMH 768 QAWNERGLWT 769 QWDTRGLWVA 770 WNVHGIWQE771 SWDTRGLWVE 772 DWDTRGLWVA 773 SWGRDGLWIE 774 EWTDNGLWAL 775SWDEKGLWSA 776 SWDSSGLWMD 777

TABLE 13 Mdm/hdm antagonist peptide sequences SEQ ID Sequence/structureNO: TFSDLW 778 QETFSDLWKLLP 779 QPTFSDLWKLLP 780 QETFSDYWKLLP 781QPTFSDYWKLLP 782 MPRFMDYWEGLN 783 VQNFIDYWTQQF 784 TGPAFTHYWATF 785IDRAPTFRDHWFALV 786 PRPALVFADYWETLY 787 PAFSRFWSDLSAGAH 788PAFSRFWSKLSAGAH 789 PXFXDYWXXL 790 QETFSDLWKLLP 791 QPTFSDLWKLLP 792QETFSDYWKLLP 793 QPTFSDYWKLLP 794

TABLE 14 Calmodulin antagonist peptide sequences SEQ IDSequence/structure NO: SCVKWGKKEFCGS 795 SCWKYWGKECGS 796 SCYEWGKLRWCGS797 SCLRWGKWSNCGS 798 SCWRWGKYQICGS 799 SCVSWGALKLCGS 800 SCIRWGQNTFCGS801 SCWQWGNLKICGS 802 SCVRWGQLSICGS 803 LKKFNARRKLKGAILTTMLAK 804RRWKKNFIAVSAANRFKK 805 RKWQKTGHAVRAIGRLSS 806 INLKALAALAKKIL 807KIWSILAPLGTTLVKLVA 808 LKKLLKLLKKLLKL 809 LKWKKLLKLLKKLLKKLL 810AEWPSLTEIKTLSHFSV 811 AEWPSPTRVISTTYFGS 812 AELAHWPPVKTVLRSFT 813AEGSWLQLLNLMKQMNN 814 AEWPSLTEIK 815

TABLE 15 Mast cell antagonists/Mast cell protease inhibitor peptidesequences SEQ ID Sequence/structure NO: SGSGVLKRPLPILPVTR 816RWLSSRPLPPLPLPPRT 817 GSGSYDTLALPSLPLHPMSS 818 GSGSYDTRALPSLPLHPMSS 819GSGSSGVTMYPKLPPHWSMA 820 GSGSSGVRMYPKLPPHWSMA 821 GSGSSSMRMVPTIPGSAKHG822 RNR NR QT NR RQK NR NRQ NR RQK NR RNRQKT 823 RNRQ 824 RNRQK 825NRQKT 826 RQKT 827

TABLE 16 SH3 antagonist peptide sequences SEQ ID Sequence/structure NO:RPLPPLP 828 RELPPLP 829 SPLPPLP 830 GPLPPLP 831 RPLPIPP 832 RPLPIPP 833RRLPPTP 834 RQLPPTP 835 RPLPSRP 836 RPLPTRP 837 SRLPPLP 838 RALPSPP 839RRLPRTP 840 RPVPPIT 841 ILAPPVP 842 RPLPMLP 843 RPLPILP 844 RPLPSLP 845RPLPSLP 846 RPLPMIP 847 RPLPLIP 848 RPLPPTP 849 RSLPPLP 850 RPQPPPP 851RQLPIPP 852 XXXRPLPPLPXP 853 XXXRPLPPIPXX 854 XXXRPLPPLPXX 855RXXRPLPPLPXP 856 RXXRPLPPLPPP 857 PPPYPPPPIPXX 858 PPPYPPPPVPXX 859LXXRPLPXΨP 860 ΨXXRPLPXLP 861 PPXΘXPPPΨP 862 +PPΨPXKPXWL 863 RPXΨPΨR+SXP864 PPVPPRPXXTL 865 ΨPΨLPΨK 866 +ΘDXPLPXLP 867

TABLE 17 Somatostatin or cortistatin mimetic peptide sequences SEQ IDSequence/structure NO: X¹-X²-Asn-Phe-Phe-Trp-Lys-Thr-Phe-X³-Ser-X⁴ 868Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr 869 Phe Ser Ser Cys LysMet Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser 870 Ser Cys Lys Cys ArgAsn Phe Phe Trp Lys Thr Phe Ser Ser Cys 871 Lys Asp Arg Met Pro Cys ArgAsn Phe Phe Trp Lys Thr 872 Phe Ser Ser Cys Met Pro Cys Arg Asn Phe PheTrp Lys Thr Phe Ser 873 Ser Cys Cys Arg Asn Phe Phe Trp Lys Thr Phe SerSer Cys 874 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr 875 Phe SerSer Cys Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser 876 Ser Cys LysCys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 877 Lys Asp Arg Met ProCys Lys Asn Phe Phe Trp Lys Thr 878 Phe Ser Ser Cys Met Pro Cys Lys AsnPhe Phe Trp Lys Thr Phe Ser 879 Ser Cys Cys Lys Asn Phe Phe Trp Lys ThrPhe Ser Ser Cys 880 Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr 881Phe Thr Ser Cys Lys Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr 882Ser Cys Lys Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 883 Lys AspArg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr 884 Phe Thr Ser Cys Met ProCys Arg Asn Phe Phe Trp Lys Thr Phe Thr 885 Ser Cys Cys Arg Asn Phe PheTrp Lys Thr Phe Thr Ser Cys 886 Asp Arg Met Pro Cys Lys Asn Phe Phe TrpLys Thr 887 Phe Thr Ser Cys Lys Met Pro Cys Lys Asn Phe Phe Trp Lys ThrPhe Thr 888 Ser Cys Lys Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys889 Lys Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr 890 Phe Thr SerCys Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr 891 Ser Cys Cys LysAsn Phe Phe Trp Lys Thr Phe Thr Ser Cys 892

TABLE 18 UKR antagonist peptide sequences SEQ ID Sequence/structure NO:AEPMPHSLNFSQYLWYT 893 AEHTYSSLWDTYSPLAF 894 AELDLWMRHYPLSFSNR 895AESSLWTRYAWPSMPSY 896 AEWHPGLSFGSYLWSKT 897 AEPALLNWSFFFNPGLH 898AEWSFYNLHLPEPQTIF 899 AEPLDLWSLYSLPPLAM 900 AEPTLWQLYQFPLRLSG 901AEISFSELMWLRSTPAF 902 AELSEADLWTTWFGMGS 903 AESSLWRIFSPSALMMS 904AESLPTLTSILWGKESV 905 AETLFMDLWHDKHILLT 906 AEILNFPLWHEPLWSTE 907AESQTGTLNTLFWNTLR 908 AEPVYQYELDSYLRSYY 909 AELDLSTFYDIQYLLRT 910AEFFKLGPNGYVYLHSA 911 FKLXXXGYVYL 912 AESTYHHLSLGYMYTLN 913 YHXLXXGYMYT914

TABLE 19 Macrophage and/or T-cell inhibiting peptide sequences SEQ IDSequence/structure NO: Xaa-Yaa-Arg NR Arg-Yaa-Xaa NR Xaa-Arg-Yaa NRYaa-Arg-Xaa NR Ala-Arg NR Arg-Arg NR Asn-Arg NR Asp-Arg NR Cys-Arg NRGln-Arg NR Glu-Arg NR Gly-Arg NR His-arg NR Ile-Arg NR Leu-Arg NRLys-Arg NR Met-Arg NR Phe-Arg NR Ser-Arg NR Thr-Arg NR Trp-Arg NRTyr-Arg NR Val-Arg NR Ala-Glu-Arg NR Arg-Glu-Arg NR Asn-Glu-Arg NRAsp-Glu-Arg NR Cys-Glu-Arg NR Gln-Glu-Arg NR Glu-Glu-Arg NR Gly-Glu-ArgNR His-Glu-Arg NR Ile-Glu-Arg NR Leu-Glu-Arg NR Lys-Glu-Arg NRMet-Glu-Arg NR Phe-Glu-Arg NR Pro-Glu-Arg NR Ser-Glu-Arg NR Thr-Glu-ArgNR Trp-Glu-Arg NR Tyr-Glu-Arg NR Val-Glu-Arg NR Arg-Ala NR Arg-Asp NRArg-Cys NR Arg-Gln NR Arg-Glu NR Arg-Gly NR Arg-His NR Arg-Ile NRArg-Leu NR Arg-Lys NR Arg-Met NR Arg-Phe NR Arg-Pro NR Arg-Ser NRArg-Thr NR Arg-Trp NR Arg-Tyr NR Arg-Val NR Arg-Glu-Ala NR Arg-Glu-AsnNR Arg-Glu-Asp NR Arg-Glu-Cys NR Arg-Glu-Gln NR Arg-Glu-Glu NRArg-Glu-Gly NR Arg-Glu-His NR Arg-Glu-Ile NR Arg-Glu-Leu NR Arg-Glu-LysNR Arg-Glu-Met NR Arg-Glu-Phe NR Arg-Glu-Pro NR Arg-Glu-Ser NRArg-Glu-Thr NR Arg-Glu-Trp NR Arg-Glu-Tyr NR Arg-Glu-Val NR Ala-Arg-GluNR Arg-Arg-Glu NR Asn-Arg-Glu NR Asp-Arg-Glu NR Cys-Arg-Glu NRGln-Arg-Glu NR Glu-Arg-Glu NR Gly-Arg-Glu NR His-Arg-Glu NR IIe-Arg-GluNR Leu-Arg-Glu NR Lys-Arg-Glu NR Met-Arg-Glu NR Phe-Arg-Glu NRPro-Arg-Glu NR Ser-Arg-Glu NR Thr-Arg-Glu NR Trp-Arg-Glu NR Tyr-Arg-GluNR Val-Arg-Glu NR Glu-Arg-Ala, NR Glu-Arg-Arg NR Glu-Arg-Asn NRGlu-Arg-Asp NR Glu-Arg-Cys NR Glu-Arg-Gln NR Glu-Arg-Gly NR Glu-Arg-HisNR Glu-Arg-Ile NR Glu-Arg-Leu NR Glu-Arg-Lys NR Glu-Arg-Met NRGlu-Arg-Phe NR Glu-Arg-Pro NR Glu-Arg-Ser NR Glu-Arg-Thr NR Glu-Arg-TrpNR Glu-Arg-Tyr NR Glu-Arg-Val NR

TABLE 20 Additional Exemplary Pharmacologically Active Peptides SEQ IDSequence/structure NO: Activity VEPNCDIHVMWEWECFERL 915 VEGF-antagonistGERWCFDGPLTWVCGEES 916 VEGF-antagonist RGWVEICVADDNGMCVTEAQ 917VEGF-antagonist GWDECDVARMWEWECFAGV 918 VEGF-antagonistGERWCFDGPRAWVCGWEI 919 VEGF-antagonist EELWCFDGPRAWVCGYVK 920VEGF-antagonist RGWVEICAADDYGRCLTEAQ 921 VEGF-antagonistRGWVEICESDVWGRCL 922 VEGF-antagonist RGWVEICESDVWGRCL 923VEGF-antagonist GGNECDIARMWEWECFERL 924 VEGF-antagonist RGWVEICAADDYGRCL925 VEGF-antagonist CTTHWGFTLC 926 MMP inhibitor CLRSGXGC 927 MMPinhibitor CXXHWGFXXC 928 MMP inhibitor CXPXC 929 MMP inhibitorCRRHWGFEFC 930 MMP inhibitor STTHWGFTLS 931 MMP inhibitor CSLHWGFWWC 932CTLA4-mimetic GFVCSGIFAVGVGRC 933 CTLA4-mimetic APGVRLGCAVLGRYC 934CTLA4-mimetic LLGRMK 935 Antiviral (HBV) ICVVQDWGHHRCTAGHMANLTSHASAI 936C3b antagonist ICVVQDWGHHRCT 937 C3b antagonist CVVQDWGHHAC 938 C3bantagonist STGGFDDVYDWARGVSSALTTTLVATR 939 Vinculin-bindingSTGGFDDVYDWARRVSSALTTTLVATR 940 Vinculin-bindingSRGVNFSEWLYDMSAAMKEASNVFPSRRS 941 Vinculin-binding RSSQNWDMEAGVEDLTAAMLGLLSTIHSSS 942 Vinculin-binding RSSPSLYTQFLVNYESAATRIQDLLIASRP 943 Vinculin-binding SRSSTGWVDLLGALQRAADATRTSIPPSLQN 944 Vinculin-binding SR DVYTKKELIECARRVSEK945 Vinculin-binding EKGSYYPGSGIAQFHIDYNNVS 946 C4BP-bindingSGIAQFHIDYNNVSSAEGWHVN 947 C4BP-binding LVTVEKGSYYPGSGIAQFHIDYNNVSSAE948 C4BP-binding GWHVN SGIAQFHIDYNNVS 949 C4BP-binding LLGRMK 950anti-HBV ALLGRMKG 951 anti-HBV LDPAFR 952 anti-HBV CXXRGDC 953Inhibition of platelet aggregation RPLPPLP 954 Src antagonist PPVPPR 955Src antagonist XFXDXWXXLXX 956 Anti-cancer (particularly for sarcomas)KACRRLFGPVDSEQLSRDCD 957 p16-mimetic RERWNFDFVTETPLEGDFAW 958p16-mimetic KRRQTSMTDFYHSKRRLIFS 959 p16-mimetic TSMTDFYHSKRRLIFSKRKP960 p16-mimetic RRLIF 961 p16-mimetic KRRQTSATDFYHSKRRLIFSRQIKIWFQN 962p16-mimetic RRMKWKK KRRLLFSKRQLKIWFQNRRMKWKK 963 p16-mimetic Asn Gln GlyArg His Phe Cys 964 CAP37 mimetic/LPS Gly Gly Ala Leu Ile His Alabinding Arg Phe Val Met Thr Ala Ala Ser Cys Phe Gln Arg His Phe Cys GiyGly Ala 965 CAP37 mimetic/LPS Leu Ile His Ala Arg Phe Val binding MetThr Ala Ala Ser Cys Gly Thr Arg Cys Gln Val Ala 966 CAP37 mimetic/LPSGly Trp Gly Ser Gln Arg Ser binding Gly Gly Arg Leu Ser Arg Phe Pro ArgPhe Val Asn Val WHWRHRIPLQLAAGR 967 carbohydrate (GDI alpha) mimeticLKTPRV 968 β2GPI Ab binding NTLKTPRV 969 β2GPI Ab binding NTLKTPRVGGC970 β2GPI Ab binding KDKATF 971 β2GPI Ab binding KDKATFGCHD 972 β2GPI Abbinding KDKATFGCHDGC 973 β2GPI Ab binding TLRVYK 974 β2GPI Ab bindingATLRVYKGG 975 β2GPI Ab binding CATLRVYKGG 976 β2GPI Ab bindingINLKALAALAKKIL 977 Membrane- transporting GWT NR Membrane- transportingGWTLNSAGYLLG 978 Membrane- transporting GWTLNSAGYLLGKINLKALAALAKKIL 979Membrane- transporting CVHAYRS 980 Antiproliferative, antiviral CVHAYRA981 Antiproliferative, antiviral CVHAPRS 982 Antiproliferative,antiviral CVHAPRA 983 Antiproiiferative, antiviral CVHSYRS 984Antiproliferative, antiviral CVHSYRA 985 Antiproliferative, antiviralCVHSPRS 986 Antiproliferative, antiviral CVHSPRA 987 Antiproliferative,antiviral CVHTYRS 988 Antiproliferative, antiviral CVHTYRA 989Antiproliferative, antiviral CVHTPRS 990 Antiproliferative, antiviralCVHTPRA 991 Antiproliferative, antiviral HWAWFK 992 anti-ischemic,growth hormone- liberating

TABLE 21 MYOSTATIN INHIBITOR PEPTIDES SEQ PEPTIBODY NAME ID PEPTIDESEQUENCE Myostatin-TN8-Con1 1036 KDKCKMWHWMCKPP Myostatin-TN8-Con2 1037KDLCAMWHWMCKPP Myostatin-TN8-Con3 1038 KDLCKMWKWMCKPP Myostatin-TN8-Con41039 KDLCKMWHWMCKPK Myostatin-TN8-Con5 1040 WYPCYEFHFWCYDLMyostatin-TN8-Con6 1041 WYPCYEGHFWCYDL Myostatin-TN8-Con7 1042IFGCKWWDVQCYQF Myostatin-TN8-Con8 1043 IFGCKWWDVDCYQF Myostatin-TN8-Con91044 ADWCVSPNWFCMVM Myostatin-TN8-Con10 1045 HKFCPWWALFCWDFMyostatin-TN8-1 1046 KDLCKMWHWMCKPP Myostatin-TN8-2 1047 IDKCAIWGWMCPPLMyostatin-TN8-3 1048 WYPCGEFGMWCLNV Myostatin-TN8-4 1049 WFTCLWNCDNEMyostatin-TN8-5 1050 HTPCPWFAPLCVEW Myostatin-TN8-6 1051 KEWCWRWKWMCKPEMyostatin-TN8-7 1052 FETCPSWAYFCLDI Myostatin-TN8-8 1053 AYKCEANDWGCWWLMyostatin-TN8-9 1054 NSWCEDQWHRCWWL Myostatin-TN8-10 1055 WSACYAGHFWCYDLMyostatin-TN8-11 1056 ANWCVSPNWFCMVM Myostatin-TN8-12 1057WTECYQQEFWCWNL Myostatin-TN8-13 1058 ENTCERWKWMCPPK Myostatin-TN8-141059 WLPCHQEGFWCMNF Myostatin-TN8-15 1060 STMCSQWHWMCNPFMyostatin-TN8-16 1061 IFGCHWWDVDCYQF Myostatin-TN8-17 1062IYGCKWWDIQCYDI Myostatin-TN8-18 1063 PDWCIDPDWWCKFW Myostatin-TN8-191064 QGHCTRWPWMCPPY Myostatin-TN8-20 1065 WQECYREGFWCLQTMyostatin-TN8-21 1066 WFDCYGPGFKCWSP Myostatin-TN8-22 1067GVRCPKGHLWCLYP Myostatin-TN8-23 1068 HWACGYWPWSCKWV Myostatin-TN8-241069 GPACHSPWWWCVFG Myostatin-TN8-25 1070 TTWCISPMWFCSQQMyostatin-TN8-26 1071 HKFCPPWAIFCWDF Myostatin-TN8-27 1072PDWCVSPRWYCNMW Myostatin-TN8-28 1073 VWKCHWFGMDCEPT Myostatin-TN8-291074 KKHCQIWTWMCAPK Myostatin-TN8-30 1075 WFQCGSTLFWCYNLMyostatin-TN8-31 1076 WSPCYDHYFYCYTI Myostatin-TN8-32 1077SWMCGFFKEVCMWV Myostatin-TN8-33 1078 EMLCMIHPVFCNPH Myostatin-TN8-341079 LKTCNLWPWMCPPL Myostatin-TN8-35 1080 VVGCKWYEAWCYNKMyostatin-TN8-36 1081 PIHCTQWAWMCPPT Myostatin-TN8-37 1082DSNCPWYFLSCVIF Myostatin-TN8-38 1083 HIWCNLAMMXCVEM Myostatin-TN8-391084 NLQCIYFLGKCIYF Myostatin-TN8-40 1085 AWRCMWFSDVCTPGMyostatin-TN8-41 1086 WFRCFLDADWCTSV Myostatin-TN8-42 1087EKICQMWSWMCAPP Myostatin-TN8-43 1088 WFYCHLNKSECTEP Myostatin-TN8-441089 FWRCAIGIDKCKRV Myostatin-TN8-45 1090 NLGCKWYEVWCFTYMyostatin-TN8-46 1091 IDLCNMWDGMCYPP Myostatin-TN8-47 1092EMPCNIWGWMCPPV Myostatin-TN12-1 1093 WFRCVLTGIVDWSECFGL Myostatin-TN12-21094 GFSCTFGLDEFYVDCSPF Myostatin-TN12-3 1095 LPWCHDQVNADWGFCMLWMyostatin-TN12-4 1096 YPTCSEKFWIYGQTCVLW Myostatin-TN12-5 1097LGPCPIHHGPWPQYCVYW Myostatin-TN12-6 1098 PFPCETHQISWLGHCLSFMyostatin-TN12-7 1099 HWGCEDLMWSWHPLCRRLP Myostatin-TN12-8 1100LPLCDADMMPTIGFCVAY Myostatin-TN12-9 1101 SHWCETTFWMNYAKCVHAMyostatin-TN12-10 1102 LPKCTHVPFDQGGFCLWY Myostatin-TN12-11 1103FSSCWSPVSRQDMFCVFY Myostatin-TN12-13 1104 SHKCEYSGWLQPLCYRPMyostatin-TN12-14 1105 PWWCQDNYVQHMLHCDSP Myostatin-TN12-15 1106WFRCMLMNSFDAFQCVSY Myostatin-TN12-16 1107 PDACRDQPWYMFMGCMLGMyostatin-TN12-17 1108 FLACFVEFELCFDS Myostatin-TN12-18 1109SAYCIITESDPYVLCVPL Myostatin-TN12-19 1110 PSICESYSTMWLPMCQHNMyostatin-TN12-20 1111 WLDCHDDSWAWTKMCRSH Myostatin-TN12-21 1112YLNCVMMNTSPFVECVFN Myostatin-TN12-22 1113 YPWCDGFMIQQGITCMFYMyostatin-TN12-23 1114 FDYCTWLNGFKDWKCWSR Myostatin-TN12-24 1115LPLCNLKEISHVQACVLF Myostatin-TN12-25 1116 SPECAFARWLGIEQCQRDMyostatin-TN12-26 1117 YPQCFNLHLLEWTECDWF Myostatin-TN12-27 1118RWRCEIYDSEFLPKCWFF Myostatin-TN12-28 1119 LVGCDNVWHRCKLFMyostatin-TN12-29 1120 AGWCHVWGEMFGMGCSAL Myostatin-TN12-30 1121HHECEWMARWMSLDCVGL Myostatin-TN12-31 1122 FPMCGIAGMKDFDFCVWYMyostatin-TN12-32 1123 RDDCTFWPEWLWKLCERP Myostatin-TN12-33 1124YNFCSYLFGVSKEACQLP Myostatin-TN12-34 1125 AHWCEQGPWRYGNICMAYMyostatin-TN12-35 1126 NLVCGKISAWGDEACARA Myostatin-TN12-36 1127HNVCTIMGPSMKWFCWND Myostatin-TN12-37 1128 NDLCAMWGWRNTIWCQNSMyostatin-TN12-38 1129 PPFCQNDNDMLQSLCKLL Myostatin-TN12-39 1130WYDCNVPNELLSGLCRLF Myostatin-TN12-40 1131 YGDCDQNHWMWPFTCLSLMyostatin-TN12-41 1132 GWMCHFDLHDWGATCQPD Myostatin-TN12-42 1133YFHCMFGGHEFEVHCESF Myostatin-TN12-43 1134 AYWCWHGQCVRFMyostatin-Linear-1 1135 SEHWTFTDWDGNEWWVRPF Myostatin-Linear-2 1136MEMLDSLFELLKDMVPISKA Myostatin-Linear-3 1137 SPPEEALMEWLGWQYGKFTMyostatin-Linear-4 1138 SPENLLNDLYILMTKQEWYG Myostatin-Linear-5 1139FHWEEGIPFHVVTPYSYDRM Myostatin-Linear-6 1140 KRLLEQFMNDLAELVSGHSMyostatin-Linear-7 1141 DTRDALFQEFYEFVRSRLVI Myostatin-Linear-8 1142RMSAAPRPLTYRDIMDQYWH Myostatin-Linear-9 1143 NDKAHFFEMFMFDVHNFVESMyostatin-Linear-10 1144 QTQAQKIDGLWELLQSIRNQ Myostatin-Linear-11 1145MLSEFEEFLGNLVHRQEA Myostatin-Linear-12 1146 YTPKMGSEWTSFWHNRIHYLMyostatin-Linear-13 1147 LNDTLLRELKMVLNSLSDMK Myostatin-Linear-14 1148FDVERDLMRWLEGFMQSAAT Myostatin-Linear-15 1149 HHGWNYLRKGSAPQWFEAWVMyostatin-Linear-16 1150 VESLHQLQMWLDQKLASGPH Myostatin-Linear-17 1151RATLLKDFWQLVEGYGDN Myostatin-Linear-18 1152 EELLREFYRFVSAFDYMyostatin-Linear-19 1153 GLLDEFSHFIAEQFYQMPGG Myostatin-Linear-20 1154YREMSMLEGLLDVLERLQHY Myostatin-Linear-21 1155 HNSSQMLLSELIMLVGSMMQMyostatin-Linear-22 1156 WREHFLNSDYIRDKLIAIDG Myostatin-Linear-23 1157QFPFYVFDDLPAQLEYWIA Myostatin-Linear-24 1158 EFFHWLHNHRSEVNHWLDMNMyostatin-Linear-25 1159 EALFQNFFRDVLTLSEREY Myostatin-Linear-26 1160QYWEQQWMTYFRENGLHVQY Myostatin-Linear-27 1161 NQRMMLEDLWRIMTPMFGRSMyostatin-Linear-29 1162 FLDELKAELSRHYALDDLDE Myostatin-Linear-30 1163GKLIEGLLNELMQLETFMPD Myostatin-Linear-31 1164 ILLLDEYKKDWKSWFMyostatin-2xTN8-19 kc 1165 QGHCTRWPWMCPPYGSGSATG GSGSTASSGSGSATGQGHCTRWPWMCPPY Myostatin-2xTN8-con6 1166 WYPCYEGHFWCYDLGSGSTASSGSGSATGWYPCYEGHFWCYD L Myostatin-2xTN8-5 kc 1167 HTPCPWFAPLCVEWGSGSATGGSGSTASSGSGSATGHTPCPW FAPLCVEW Myostatin-2xTN8-18 kc 1168PDWCIDPDWWCKFWGSGSATG GSGSTASSGSGSATGPDWCID PDWWCKFW Myostatin-2xTN8-11kc 1169 ANWCVSPNWFCMVMGSGSATG GSGSTASSGSGSATGANWCVS PNWFCMVMMyostatin-2xTN8-25 kc 1170 PDWCIDPDWWCKFWGSGSATG GSGSTASSGSGSATGPDWCIDPDWWCKFW Myostatin-2xTN8-23 kc 1171 HWACGYWPWSCKWVGSGSATGGSGSTASSGSGSATGHWACGY WPWSCKWV Myostatin-TN8-29-19 kc 1172KKHCQIWTWMCAPKGSGSATG GSGSTASSGSGSATGQGHCTR WPWMCPPY Myostatin-TN8-19-29kc 1173 QGHCTRWPWMCPPYGSGSATG GSGSTASSGSGSATGKKHCQI WTWMCAPKMyostatin-TN8-29-19 kn 1174 KKHCQIWTWMCAPKGSGSATG GSGSTASSGSGSATGQGHCTRWPWMCPPY Myostatin-TN8-29-19-8 g 1175 KKHCQIWTWMCAPKGGGGGGGGQGHCTRWPWMCPPY Myostatin-TN8-19-29-6 gc 1176 QGHCTRWPWMCPPYGGGGGGKKHCQIWTWMCAPK

TABLE 22 MYOSTATIN INHIBITOR PEPTIDES Affinity- SEQ matured ID peptibodyNO: Peptide sequence mTN8-19-1 1177 VALHGQCTRWPWMCPPQREG mTN8-19-2 1178YPEQGLCTRWPWMCPPQTLA mTN8-19-3 1179 GLNQGHCTRWPWMCPPQDSN mTN8-19-4 1180MITQGQCTRWPWMCPPQPSG mTN8-19-5 1181 AGAQEHCTRWPWMCAPNDWI mTN8-19-6 1182GVNQGQCTRWRWMCPPNGWE mTN8-19-7 1183 LADHGQCIRWPWMCPPEGWE mTN8-19-8 1184ILEQAQCTRWPWMCPPQRGG mTN8-19-9 1185 TQTHAQCTRWPWMCPPQWEG mTN8-19-10 1186VVTQGHCTLWPWMCPPQRWR mTN8-19-11 1187 IYPHDQCTRWPWMCPPQPYP mTN8-19-121188 SYWQGQCTRWPWMCPPQWRG mTN8-19-13 1189 MWQQGHCTRWPWMCPPQGWGmTN8-19-14 1190 EFTQWHCTRWPWMCPPQRSQ mTN8-19-15 1191LDDQWQCTRWPWMCPPQGFS mTN8-19-16 1192 YQTQGLCTRWPWMCPPQSQR mTN8-19-171193 ESNQGQCTRWPWMCPPQGGW mTN8-19-18 1194 WTDRGPCTRWPWMCPPQANGmTN8-19-19 1195 VGTQGQCTRWPWMCPPYETG mTN8-19-20 1196PYEQGKCTRWPWMCPPYEVE mTN8-19-21 1197 SEYQGLCTRWPWMCPPQGWK mTN8-19-221198 TFSQGHCTRWPWMCPPQGWG mTN8-19-23 1199 PGAHDHCTRWPWMCPPQSRYmTN8-19-24 1200 VAEEWHCRRWPWMCPPQDWR mTN8-19-25 1201VGTQGHCTRWPWMCPPQPAG mTN8-19-26 1202 EEDQAHCRSWPWMCPPQGWV mTN8-19-271203 ADTQGHCTRWPWMCPPQHWF mTN8-19-28 1204 SGPQGHCTRWPWMCAPQGWFmTN8-19-29 1205 TLVQGHCTRWPWMCPPQRWV mTN8-19-30 1206GMAHGKCTRWAWMCPPQSWK mTN8-19-31 1207 ELYHGQCTRWPWMCPPQSWA mTN8-19-321208 VADHGHCTRWPWMCPPQGWG mTN8-19-33 1209 PESQGHCTRWPWMCPPQGWGmTN8-19-34 1210 IPAHGHCTRWPWMCPPQRWR mTN8-19-35 1211FTVHGHCTRWPWMCPPYGWV mTN8-19-36 1212 PDFPGHCTRWRWMCPPQGWE mTN8-19-371213 QLWQGPCTQWPWMCPPKGRY mTN8-19-38 1214 HANDGHCTRWQWMCPPQWGGmTN8-19-39 1215 ETDHGLCTRWPWMCPPYGAR mTN8-19-40 1216GTWQGLCTRWPWMCPPQGWQ mTN8-19 con1 1217 VATQGQCTRWPWMCPPQGWG mTN8-19 con21218 VATQGQCTRWPWMCPPQRWG mTN8 con6-1 1219 QREWYPCYGGHLWCYDLHKA mTN8con6-2 1220 ISAWYSCYAGHFWCWDLKQK mTN8 con6-3 1221 WTGWYQCYGGHLWCYDLRRKmTN8 con6-4 1222 KTFWYPCYDGHFWCYNLKSS mTN8 con6-5 1223ESRWYPCYEGHLWCFDLTET

TABLE 23 MYOSTATIN INHIBITOR PEPTIDES Affinity SEQ matured ID peptibodyNO: Peptide Sequence L2 1224 MEMLDSLFELLKDMVPISKA mL2-Con1 1225RMEMLESLLELLKEIVPMSKAG mL2-Con2 1226 RMEMLESLLELLKEIVPMSKAR mL2-1 1227RMEMLESLLELLKDIVPMSKPS mL2-2 1228 GMEMLESLFELLQEIVPMSKAP mL2-3 1229RMEMLESLLELLKDIVPISNPP mL2-4 1230 RIEMLESLLELLQEIVPISKAE mL2-5 1231RMEMLQSLLELLKDIVPMSNAR mL2-6 1232 RMEMLESLLELLKEIVPTSNGT mL2-7 1233RMEMLESLFELLKEIVPMSKAG mL2-8 1234 RMEMLGSLLELLKEIVPMSKAR mL2-9 1235QMELLDSLFELLKEIVPKSQPA mL2-10 1236 RMEMLDSLLELLKEIVPMSNAR mL2-11 1237RMEMLESLLELLHEIVPMSQAG mL2-12 1238 QMEMLESLLQLLKEIVPMSKAS mL2-13 1239RMEMLDSLLELLKDMVPMTTGA mL2-14 1240 RIEMLESLLELLKDMVPMANAS mL2-15 1241RMEMLESLLQLLNEIVPMSRAR mL2-16 1242 RMEMLESLFDLLKELVPMSKGV mL2-17 1243RIEMLESLLELLKDIVPIQKAR mL2-18 1244 RMELLESLFELLKDMVPMSDSS mL2-19 1245RMEMLESLLEVLQEIVPRAKGA mL2-20 1246 RMEMLDSLLQLLNEIVPMSHAR mL2-21 1247RMEMLESLLELLKDIVPMSNAG mL2-22 1248 RMEMLQSLFELLKGMVPISKAG mL2-23 1249RMEMLESLLELLKEIVPNSTAA mL2-24 1250 RMEMLQSLLELLKEIVPISKAG mL2-25 1251RIEMLDSLLELLNELVPMSKAR L-15 1252 HHGWNYLRKGSAPQWFEAWV mL15-con1 1253QVESLQQLLMWLDQKLASGPQG mL15-1 1254 RMELLESLFELLKEMVPRSKAV mL15-2 1255QAVSLQHLLMWLDQKLASGPQH mL15-3 1256 DEDSLQQLLMWLDQKLASGPQL mL15-4 1257PVASLQQLLIWLDQKLAQGPHA mL15-5 1258 EVDELQQLLNWLDHKLASGPLQ mL15-6 1259DVESLEQLLMWLDHQLASGPHG mL15-7 1260 QVDSLQQVLLWLEHKLALGPQV mL15-8 1261GDESLQHLLMWLEQKLALGPHG mL15-9 1262 QIEMLESLLDLLRDMVPMSNAF mU15-10 1263EVDSLQQLLMWLDQKLASGPQA mL15-11 1264 EDESLQQLLIYLDKMLSSGPQV mL15-12 1265AMDQLHQLLIWLDHKLASGPQA mL15-13 1266 RIEMLESLLELLDEIALIPKAW mL15-14 1267EVVSLQHLLMWLEHKLASGPDG mL15-15 1268 GGESLQQLLMWLDQQLASGPQR mL15-16 1269GVESLQQLLIFLDHMLVSGPHD mL15-17 1270 NVESLEHLMMWLERLLASGPYA mL15-18 1271QVDSLQQLLIWLDHQLASGPKR mL15-19 1272 EVESLQQLLMWLEHKLAQGPQG mL15-20 1273EVDSLQQLLMWLDQKLASGPHA mL15-21 1274 EVDSLQQLLMWLDQQLASGPQK mL15-22 1275GVEQLPQLLMWLEQKLASGPQR mL15-23 1276 GEDSLQQLLMWLDQQLAAGPQV mL15-24 1277ADDSLQQLLMWLDRKLASGPHV mL15-25 1278 PVDSLQQLLIWLDQKLASGPQG L-17 1279RATLLKDFWQLVEGYGDN mL17-con1 1280 DWRATLLKEFWQLVEGLGDNLV mL17-con2 1281QSRATLLKEFWQLVEGLGDKQA mL17-1 1282 DGRATLLTEFWQLVQGLGQKEA mL17-2 1283LARATLLKEFWQLVEGLGEKVV mL17-3 1284 GSRDTLLKEFWQLVVGLGDMQT mL17-4 1285DARATLLKEFWQLVDAYGDRMV mL17-5 1286 NDRAQLLRDFWQLVDGLGVKSW mL17-6 1287GVRETLLYELWYLLKGLGANQG mL17-7 1288 QARATLLKEFCQLVGCQGDKLS mL17-8 1289QERATLLKEFWQLVAGLGQNMR mL17-9 1290 SGRATLLKEFWQLVQGLGEYRW mL17-10 1291TMRATLLKEFWLFVDGQREMQW mL17-11 1292 GERATLLNDFWQLVDGQGDNTG mL17-12 1293DERETLLKEFWQLVHGWGDNVA mL17-13 1294 GGRATLLKELWQLLEGQGANLV mL17-14 1295TARATLLNELVQLVKGYGDKLV mL17-15 1295 GMRATLLQEFWQLVGGQGDNWM mL17-16 1297STRATLLNDLWQLMKGWAEDRG mL17-17 1298 SERATLLKELWQLVGGWGDNFG mL17-18 1299VGRATLLKEFWQLVEGLVGQSR mL17-19 1300 EIRATLLKEFWQLVDEWREQPN mE17-20 1301QLRATLLKEFLQLVHGLGETDS mL17-21 1302 TQRATLLKEFWQLIEGLGGKHV mL17-22 1303HYRATLLKEFWQLVDGLREQGV mL17-23 1304 QSRVTLLREFWQLVESYRPIVN mL17-24 1305LSRATLLNEFWQFVDGQRDKRM mE17-25 1306 WDRATLLNDFWHLMEELSQKPG mL17-26 1307QERATLLKEFWRMVEGLGKNRG mL17-27 1308 NERATLLREFWQLVGGYGVNQR L-20 1309YREMSMLEGLLDVLERLQHY mL20-1 1310 HQRDMSMLWELLDVLDGLRQYS mL20-2 1311TQRDMSMLDGLLEVLDQLRQQR mL20-3 1312 TSRDMSLLWELLEELDRLGHQR mL20-4 1313MQHDMSMLYGLVELLESLGHQI mL20-5 1314 WNRDMRMLESLFEVLDGLRQQV mL20-6 1315GYRDMSMLEGLLAVLDRLGPQL mL20 con1 1316 TQRDMSMLEGLLEVLDRLGQQR mL20 con21317 WYRDMSMLEGLLEVLDRLGQQR L-21 1318 HNSSQMLLSELIMLVGSMMQ mL21-1 1319TQNSRQMLLSDFMMLVGSMIQG mL21-2 1320 MQTSRHILLSEFMMLVGSIMHG mL21-3 1321HDNSRQMLLSDLLHLVGTMIQG mL21-4 1322 MENSRQNLLRELIMLVGNMSHQ mL21-5 1323QDTSRHMLLREFMMLVGEMIQG mL21 con1 1324 DQNSRQMLLSDLMILVGSMIQG L-24 1325EFFHWLHNHRSEVNHWLDMN mL24-1 1326 NVFFQWVQKHGRVVYQWLDINV mL24-2 1327FDFLQWLQNHRSEVEHWLVMDV

TABLE 24 MYOSTATIN INHIBITOR PEPTIDES Peptibody Name Peptide 2xmTN8-Con6- M-GAQ-WYPCYEGHFWCYDL- (N)-1 K GSGSATGGSGSTASSGSGSATG-WYPCYEGHFWCYDL-LE-5G-FC (SEQ ID NO: 1328) 2x mTN8-Con6-FC-5G-AQ-WYPCYEGHFWCYDL- (C)-1 K GSGSATGGSGSTASSGSGSATG-WYPCYEGHFWCYDL-LE (SEQ ID NO: 1329) 2x mTN8-Con7- M-GAQ-IFGCKWWDVQCYQF-(N)-1 K GSGSATGGSGSTASSGSGSATG- IFGCKWWDVQCYQF-LE-5G-FC (SEQ ID NO:1330) 2x mTN8-Con7- FC-5G-AQ-IFGCKWWDVQCYQF- (C)-1 KGSGSATGGSGSTASSGSGSATG- IFGCKWWDVQCYQF-LE (SEQ ID NO: 1331) 2xmTN8-Con8- M-GAQ-IFGCKWWDVDCYQF (N)-1 K GSGSATGGSGSTASSGSGSATG-IFGCKWWDVDCYQF-LE-5G-FC (SEQ ID NO: 1332) 2x mTN8-Con8-FC-5G-AQ-IFGCKWWDVDCYQF- (C)-1 K GSGSATGGSGSTASSGSGSATG-IFGCKWWDVDCYQF-LE (SEQ ID NO: 1333) 2X mTN8-19-7FC-5G-AQ-LADHGQCIRWPWMCPPEGWELEGSGSATGGSGSTASSGSGSATGLADHGQCIRWPWMCPPEGW E-LE (SEQ ID NO: 1334) 2X mTN8-19-7FC-5G-AQ-LADHGQCIRWPWMCPPEGWEGSGSATG ST-GG del2xGSGGGASSGSGSATGLADHGQCIRWPWMCPPEGWE LE (SEQ ID NO: 1335) 2X mTN8-19-21FC-5G-AQ-SEYQGLCTRWPWMCPPQGWKLEGSGSATGGSGSTASSGSGSATGSEYQGLCTRWPWMCPPQGW K-LE (SEQ ID NO: 1336) 2XmTN8-19-21 FC-5G-AQ-SEYQGLCTRWPWMCPPQGWKGSGSATG ST-GG del2xGSGGGASSGSGSATGSEYQGLCTRWPWMCPPQGWK LE (SEQ ID NO: 1337) 2X mTN8-19-22FC-5G-AQ-TFSQGHCTRWPWMCPPQGWGLEGSGSATGGSGSTASSGSGSATGTFSQGHCTRWPWMCPPQGW G-LE (SEQ ID NO: 1338) 2XmTN8-19-32 FC-5G-AQ-VADHGHCTRWPWMCPPQGWGLEGSGSATGGSGSTASSGSGSATGVADHGHCTRWPWMCPPQGW G-LE (SEQ ID NO: 1339) 2XmTN8-19-32 FC-5G-AQ-VADHGHCTRWPWMCPPQGWGGSGSATG ST-GG del2xGSGGGASSGSGSATGVADHGHCTRWPWVCPPQGWG LE (SEQ ID NO: 1340) 2X mTN8-19-33FC-5G-AQ-PESQGHCTRWIPWMCPPQGWGLEGSGSATGGSGSTASSGSGSATGPESQGHCTRWPWMCPPQG WGLE (SEQ ID NO: 1341) 2XmTN8-19-33 FC-5G-AQ-PESQGHCTRWPWMCPPQGWGGSGSATG ST-GG del2xGSGGGASSGSGSATGPESQGHCTRWPWMCP PQGWG LE (SEQ ID NO: 1342)

TABLE 25 Integrin-antagonist peptide sequences SEQ. IDSequence/structure NO: CLCRGDCIC 1344 CWDDGWLC 1345 CWDDLWWLC 1346CWDDGLMC 1347 CWDDGWMC 1348 CSWDDGWLC 1349 CPDDLWWLC 1350 NGR 1351 GSL1352 RGD 1353 CGRECPRLCQSSC 1354 CNGRCVSGCAGRC 1355 CLSGSLSC 1356 GSL1357 NGRAHA 1358 CNGRC 1359 CDCRGDCFC 1360 CGSLVRC 1361 DLXXL 1362RTDLDSLRTYTL 1363 RTDLDSLRTY 1364 RTDLDSLRT 1365 RTDLDSLR 1366GDLDLLKLRLTL 1367 GDLHSLRQLLSR 1368 RDDLHMLRLQLW 1369 SSDLHALKKRYG 1370RGDLKQLSELTW 1371 CXXRGDC 1372 STGGFDDVYDWARGVSSALTTTLVATR 1373STGGFDDVYDWARRVSSALTTTLVATR 1374 SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 1375SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 1376 SSPSLYTQFLVNYESAATRIQDLLIASRPSR 1377SSTGWVDLLGALQRAADATRTSIPPSLQNSR 1378 DVYTKKELIECARRVSEK 1379 RGDGX 1380CRGDGXC 1381 CARRLDAPC 1382 CPSRLDSPC 1383 CDCRGDCFC 1384 CDCRGDCLC 1385RGDLAALSAPPV 1386

TABLE 26 Selectin antagonist peptide sequences SEQ. IDSequence/structure NO: DITWDQLWDLMK 1387 DITWDELWKIMN 1388 DYTWFELWDMMQ1389 QITWAQLWNMMK 1390 DMTWHDLWTLMS 1391 DYSWHDLWEMMS 1392 EITWDQLWEVMN1393 HVSWEQLWDIMN 1394 HITWDQLWRIMT 1395 RNMSWLELWEHMK 1396AEWTWDQLWHVMNPAESQ 1397 HRAEWLALWEQMSP 1398 KKEDWLALWRIMSV 1399ITWDQLWDLMK 1400 DITWDQLWDLMK 1401 DITWDQLWDLMK 1402 DITWDQLWDLMK 1403CQNRYTDLVAIQNKNE 1404 AENWADNEPNNKRNNED 1405 RKNNKTWTWVGTKKALTNE 1406KKALTNEAENWAD 1407 CQXRYTDLVAIQNKXE 1408 AENWADGEPNNKXNXED 1409

TABLE 27 Vinculin binding peptides SEQ. ID Sequence/structure NO:SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 1410 SSPSLYTQFLVNYESAATRIQDLLLASRPSR 1411SSTGWVDLLGALQRAADATRTSIPPSLQNSR 1412 DVYTKKELIECARRVSEK 1413STGGFDDVYDWARGVSSALTTTLVATR 1414 STGGFDDVYDWARRVSSALTTTLVATR 1415SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 1416

TABLE 28 Laminin-related peptide sequences SEQ. ID Sequence/structureNO: YIGSRYIGSR [i.e., (YIGSR)₂] 1417 YLGSRYIGSRYIGSR [i.e., (YIGSR)₃]1418 YIGSRYIGSRYIGSRYIGSR [i.e., (YIGSR)₄] 1419YIGSRYIGSRYIGSRYIGSRYIGSR [i.e., (YIGSR)₅] 1420 IPCNNKGAHSVGLMWWMLAR1421 YIGSRREDVEILDVPDSGR 1422 RGDRGDYIGSRRGD 1423YIGSRYIGSRYIGSRYIGSRYIGSR 1424 REDVEILDVYIGSRPDSGR 1425YIGSRREDVEILDVPDSGR 1426

TABLE 29 NGF Modulating Peptides SEQ ID Sequence of Peptide Portion ofNO: Fc-Peptide Fusion Product 1427 TGYTEYTEEWPMGFGYQWSF 1428TDWLSDFPFYEQYFGLMPPG 1429 FMRFPNPWKLVEPPQGWYYG 1430 VVKAPHFEFLAPPHFHEFPF1431 FSYIWIDETPSNIDRYMLWL 1432 VNFPKVPEDVEPWPWSLKLY 1433TWHPKTYEEFALPFFVPEAP 1434 WHFGTPYIQQQPGVYWLQAP 1435 VWNYGPFFMNFPDSTYFLHE1436 WRIHSKPLDYSHVWFFPADF 1437 FWDGNQPPDILVDWPWNPPV 1438FYSLEWLKDHSEFFQTVTEW 1439 QFMELLKFFNSPGDSSHHFL 1440 TNVDWISNNWEHMKSFFTED1441 PNEKPYQMQSWFPPDWPVPY 1442 WSHTEWVPQVWWKPPNHFYV 1443WGEWINDAQVHMHEGFISES 1444 VPWEHDHDLWEIISQDWHIA 1445 VLHLQDPRGWSNFPPGVLEL1446 IHGCWFTEEGCVWQ 1447 YMQCQFARDGCPQW 1448 KLQCQYSESGCPTI 1449FLQCEISGGACPAP 1450 KLQCEFSTSGCPDL 1451 KLQCEFSTQGCPDL 1452KLQCEFSTSGCPWL 1453 IQGCWFTEEGCPWQ 1454 SFDCDNPWGHVLQSCFGF 1455SFDCDNPWGHKLQSCFGF

TABLE 30 TALL MODULATING PEPTIDES SEQ ID Sequence/structure NO:LPGCKWDLLIKQWVCDPL-Λ-V1 1456 V1-Λ-LPGCKWDLLIKQWVCDPL 1457LPGCKWDLLIKQWVCDPL-Λ- 1458 LPGCKWDLLIKQWVCDPL-Λ-V1V1-Λ-LPGCKWDLLIKQWVCDPL-Λ- 1459 LPGCKWDLLLKQWVCDPLSADCYFDILTKSDVCTSS-Λ-V1 1460 V1-Λ-SADCYFDILTKSDVCTSS 1461SADCYFDILTKSDVTSS-Λ- 1462 SADCYFDILTKSDVTSS-Λ-V1V1-A-SADCYFDILTKSDVTSS-Λ- 1463 SADCYFDILTKSDVTSS FHDCKWDLLTKQWVCHGL-Λ-V11464 V1-Λ-FHDCKWDLLTKQWVCHGL 1465 FHDCKWDLLTKQWVCHGL-Λ- 1466FHDCKWDLLTKQWVCHGL-Λ-V1 V1-Λ-FHDCKWDLLTKQWVCHGL-Λ- 1467FHDCKWDLLTKQWVCHGL

TABLE 31 TALL-1 inhibitory peptibodies. Pepti- body SEQ ID Peptibody NOPeptide Sequence TALL-1-8-1 1468 MPGTCFPFPW ECTHAGGGGG VDKTHTCPPC 1-aPAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKTKPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVYTLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSKLTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-8- 1469 MWGACWPFPWECFKEGGGGG VDKTHTCPPC 2-a PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHEDPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALPAPIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV KGFYPSDIAV EWESNGQPENNYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGKTALL-1-8- 1470 MVPFCDLLTK HCFEAGGGGG VDKTHTCPPC 4-a PAPELLGGPSVFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNSTYRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELTKNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQGNVFSCSVMH EALHNHYTQK SLSLSPGK TALL-1-12- 1471 MGSRCKYKWD VLTKQCFHHGGGGGVDKTHT 4-a CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKFNWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12-1472 MLPGCKWDLL IKQWVCDPLG GGGGVDKTHT 3-a CPPCPAPELL GGPSVFLFPPKPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSVLTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSLTCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSCSVMHEALHNH YTQKSLSLSP GK TALL-1-12- 1473 MSADCYFDIL TKSDVCTSSG GGGG 5-aVDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 1474MSDDCMYDQL TRMFICSNLG GGGGVDKTHT 8-a CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP GK TALL-1-12- 1475 MDLNCKYDEL TYKEWCQFNG GGGGVDKTHT 9-aCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 1476MFHDCKYDLL TRQMVCHGLG GGGGVDKTHT 10-a CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP GK TALL-1-12- 1477 MRNHCFWDHL LKQDICPSPG GGGGVDKTHT 11-aCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1-12- 1478MANQCWWDSL TKKNVCEFFG GGGGVDKTHT 14-a CPPCPAPELL GGPSVFLFPP KPKDTLMISRTPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLNGKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNHYTQKSLSLSP GK TALL-1- 1479 MFHDCKWDLL TKQWVCHGLG GGGGVDKTHT consensusCPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPREPQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFFLYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK TALL-1 12-3 1480MLPGCKWDLL IKQWVCDPLG SGSATGGSGS tandem TASSGSGSAT HMLPGCKWDL LIKQWVCDPLdimer GGGGGVDKTH TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVKFNWYVDGVEV HNAKTKPREE QYNSTYRVYS VLTVLHQDWL NGKEYKCKVS NKALPAPIEKTISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP SDIAVEWESN GQPENNYKTTPPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHN HYTQKSLSLS PGK TALL-1 1481MFHDCKWDLL TKQWVCHGLG SGSATGGSGS consensus TASSGSGSAT HMFHDCKWDLLTKQWVCHGL tandem GGGGGVDKTH TCPPCPAPEL LGGPSVFLFP dimer PKPKDTLMISRTPEVTCVVV DVSHEDPEVK FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWLNGKEYKCKVS NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYPSDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS CSVMHEALHNHYTQKSLSLS PGK

TABLE 32 ANG-2 INHIBITOR PEPTIDES PEPTIDE SEQ ID NO. PEPTIDE SEQUENCECon4-44 1482 PIRQEECDWDPWTCEHMWEV Con4-40 1483 TNIQEECEWDPWTCDHMPGKCon4-4 1484 WYEQDACEWDPWTCEHMAEV Con4-31 1485 NRLQEVCEWDPWTCEHMENVCon4-C5 1486 AATQEECEWDPWTCEHMIRS Con4-42 1487 LRHQEGCEWDPWTCEHMFDWCon4-35 1488 VPRQKDCEWDPWTCEHMYVG Con4-43 1489 SISHEECEWDPWTCEHMQVGCon4-49 1490 WAAQEECEWDPWTCEHMGRM Con4-27 1491 TWPQDKCEWDPWTCEHMGSTCon4-48 1492 GHSQEECGWDPWTCEHMGTS Con4-46 1493 QHWQEECEWDPWTCDHMPSKCon4-41 1494 NVRQEKCEWDPWTCEHMPVR Con4-36 1495 KSGQVECNWDPWTCEHMPRNCon4-34 1496 VKTQEHCDWDPWTCEHMREW Con4-28 1497 AWGQEGCDWDPWTCEHMLPMCon4-39 1498 PVNQEDCEWDPWTCEHMPPM Con4-25 1499 RAPQEDCEWDPWTCAHMDIKCon4-50 1500 HGQNMECEWDPWTCEHMFRY Con4-38 1501 PRLQEECVWDPWTCEHMPLRCon4-29 1502 RTTQEKCEWDPWTCEHMESQ Con4-47 1503 QTSQEDCVWDPWTCDHMVSSCon4-20 1504 QVIGRPCEWDPWTCEHLEGL Con4-45 1505 WAQQEECAWDPWTCDHMVGLCon4-37 1506 LPGQEDCEWDPWTCEHMVRS Con4-33 1507 PMNQVECDWDPWTCEHMPRSAC2-Con4 1508 FGWSHGCEWDPWTCEHMGST Con4-32 1509 KSTQDDCDWDPWTCEHMVGPCon4-17 1510 GPRISTCQWDPWTCEHMDQL Con4-8 1511 STIGDMCEWDPWTCAHMQVDAC4-Con4 1512 VLGGQGCEWDPWTCRLLQGW Con4-1 1513 VLGGQGCQWDPWTCSHLEDGCon4-C1 1514 TTIGSMCEWDPWTCAHMQGG Con4-21 1515 TKGKSVCQWDPWTCSHMQSGCon4-C2 1516 TTIGSMCQWDPWTCAHMQGG Con4-18 1517 WVNEVVCEWDPWTCNHWDTPCon4-19 1518 VVQVGMCQWDPWTCKHMRLQ Con4-16 1519 AVGSQTCEWDPWTCAHLVEVCon4-11 1520 QGMKMFCEWDPWTCAHIVYR Con4-C4 1521 TTIGSMCQWDPWTCEHMQGGCon4-23 1522 TSQRVGCEWDPWTCQHLTYT Con4-15 1523 QWSWPPCEWDPWTCQTVWPSCon4-9 1524 GTSPSFCQWDPWTCSHMVQG TN8-Con4* 1525 QEECEWDPWTCEHM

TABLE 33 ANG-2 INHIBITOR PEPTIDES Peptide SEQ ID NO. Peptide SequenceL1-1 1526 QNYKPLDELDATLYEHFIFHYT L1-2 1527 LNFTPLDELEQTLYEQWTLQQS L1-31528 TKFNPLDELEQTLYEQWTLQHQ L1-4 1529 VKFKPLDALEQTLYEHWMFQQA L1-5 1530VKYKPLDELDEILYEQQTFQER L1-7 1531 TNFMPMDDLEQRLYEQFILQQG L1-9 1532SKFKPLDELEQTLYEQWTLQHA L1-10 1533 QKFQPLDELEQTLYEQFMLQQA L1-11 1534QNFKPMDELEDTLYKQFLFQHS L1-12 1535 YKFTPLDDLEQTLYEQWTLQHV L1-13 1536QEYEPLDELDETLYNQWMFHQR L1-14 1537 SNFMPLDELEQTLYEQFMLQHQ L1-15 1538QKYQPLDELDKTLYDQFMLQQG L1-16 1539 QKFQPLDELEETLYKQWTLQQR L1-17 1540VKYKPLDELDEWLYHQFTLHHQ L1-18 1541 QKFMPLDELDEILYEQFMFQQS L1-19 1542QTFQPLDDLEEYLYEQWIRRYH L1-20 1543 EDYMPLDALDAQLYEQFILLHG L1-21 1544HTFQPLDELEETLYYQWLYDQL L1-22 1545 YKFNPMDELEQTLYEEFLFQHA AC6-L1 1546TNYKPLDELDATLYEHWILQHS L1-C1 1547 QKFKPLDELEQTLYEQWTLQQR L1-C2 1548TKFQPLDELDQTLYEQWTLQQR L1-C3 1549 TNFQPLDELDQTLYEQWTLQQR L1 1550KFNPLDELEETLYEQFTFQQ

TABLE 34 ANG-2 INHIBITOR PEPTIDES Peptide SEQ ID NO. Sequence Con1-11551 AGGMRPYDGMLGWPNYDVQA Con1-2 1552 QTWDDPCMHILGPVTWRRCI Con1-3 1553APGQRPYDGMLGWPTYQRIV Con1-4 1554 SGQLRPCEEIFGCGTQNLAL Con1-5 1555FGDKRPLECMFGGPIQLCPR Con1-6 1556 GQDLRPCEDMFGCGTKDWYG Con1 1557KRPCEEIFGGCTYQ

TABLE 35 ANG-2 INHIBITOR PEPTIDES Peptide SEQ ID NO: Sequence 12-9-11558 GFEYCDGMEDPFTFGCDKQT 12-9-2 1559 KLEYCDGMEDPFTQGCDNQS 12-9-3 1560LQEWCEGVEDPFTFGCEKQR 12-9-4 1561 AQDYCEGMEDPFTFGCEMQK 12-9-5 1562LLDYCEGVQDPFTFGCENLD 12-9-6 1563 HQEYCEGMEDPFTFGCEYQG 12-9-7 1564MLDYCEGMDDPFTFGCDKQM 12-9-C2 1565 LQDYCEGVEDPFTFGCENQR 12-9-C1 1566LQDYCEGVEDPFTFGCEKQR 12-9 1567 FDYCEGVEDPFTFGCDNH

TABLE 36 Ang-2 Binding Peptides Peptide Seq Id No. Sequence TN8-8 1568KRPCEEMWGGCNYD TN8-14 1569 HQICKWDPWTCKHW TN8-Con1 1570 KRPCEEIFGGCTYQTN8-Con4 1571 QEECEWDPWTCEHM TN12-9 1572 FDYCEGVEDPFTFGCDNH L1 1573KFNPLDELEETLYEQFTFQQ C17 1574 QYGCDGFLYGCMIN

TABLE 37 Ang-2 Binding Peptides Peptibody Peptibody Sequence L1 (N)MGAQKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (SEQ ID NO: 1575) L1 (N) WTMKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (SEQ ID NO: 1576) L1 (N) 1K WTMKFNPLDELEETLYEQFTFQQGSGSATGGSGSTASS GSGSATHLEGGGGG-Fc (SEQ ID NO: 1577)2xL1 (N) MGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNP LDELEETLYEQFTFQQLEGGGGG-Fc(SEQ ID NO: 1578) 2xL1 (N) WT MKFNPLDELEETLYEQFTFQQGGGGGGGKFNPLDELEETLYEQFTFQQLEGGGGG-Fc (SEQ ID NO: 1579) Con4 (N)MGAQQEECEWDPWTCEHMLEGGGGG-Fc (SEQ ID NO: 1580) Con4 (N) 1K-WTMQEEGEWDPWTCEHMGSGSATGGSGSTASSGSGSAT HLEGGGGG-Fc (SEQ ID NO: 1581)2xCon4 (N) 1K MGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLEGGGGG-Fc (SEQ ID NO: 1582) L1 (C)M-Fc-GGGGGAQKFNPLDELEETLYEQFTFQQLE (SEQ ID NO: 1583) L1 (C) 1KM-Fc-GGGGGAQGSGSATGGSGSTASSGSGSATHKF NPLDELEETLYEQFTFQQLE (SEQ ID NO:1584) 2xL1 (C) M-Fc-GGGGGAQKFNPLDELEETLYEQFTFQQGGGGGGGGKFNPLDELEETLYEQFTFQQLE (SEQ ID NO: 1585) Con4 (C)M-Fc-GGGGGAQQEECEWDPWTCEHMLE (SEQ ID NO: 1586) Con4 (C) 1KM-Fc-GGGGGAQGSGSATGGSGSTASSGSGSATHQE ECEWDPWTCEHMLE (SEQ ID NO: 1587)2xCon4 (C) 1K M-Fc-GGGGGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE (SEQ ID NO: 1588) Con4-L1 (N)MGAQEECEWDPWTCEHMGGGGGGGGKFNPLDELEETLYEQFTFQQGSGSATGGSGSTASSGSGSATHLEGGG GG-Fc (SEQ ID NO: 1589) Con4-L1 (C)M-Fc-GGGGGAQGSGSATGGSGSTASSGSGSATHKFNPLDELEETLYEQFTFQQGGGGGQEECEWDPWTCEH MLE (SEQ ID NO: 1590) TN-12-9 (N)MGAQ-FDYCEGVEDPFTFGCDNHLE-GGGGG-Fc (SEQ ID NO: 1591) C17 (N)MGAQ-QYGCDGFLYGCMINLE-GGGGG-Fc (SEQ ID NO: 1592) TN8-8 (N)MGAQ-KRPCEEMWGGCNYDLEGGGGG-Fc (SEQ ID NO: 1593) TN8-14 (N)MGAQ-HQICKWDPWTCKHWLEGGGGG-Fc (SEQ ID NO: 1594) Con1 (N)MGAQ-KRPCEEIFGGCTYQLEGGGGG-Fc (SEQ ID NO: 1595)

TABLE 38 Ang-2 Binding Peptides Con4 Derived Affinity- Matured PbsPeptibody Sequence (Seq Id No:) Con4-44M-Fc-GGGGGAQ-PIRQEECDWDPWTCEHMWEV-LE (C) (SEQ ID NO: 1596) Con4-40M-Fc-GGGGGAQ-TNIQEECEWDPWTCDHMPGK-LE (C) (SEQ ID NO: 1597) Con4-4M-Fc-GGGGGAQ-WYEQDACEWDPWTCEHMAEV-LE (C) (SEQ ID NO: 1598) Con4-31M-Fc-GGGGGAQ-NRLQEVCEWDPWTCEHMENV-LE (C) (SEQ ID NO: 1599) Con4-C5M-Fc-GGGGGAQ-AATQEECEWDPWTCEHMPRS-LE (C) (SEQ ID NO: 1600) Con4-42M-Fc-GGGGGAQ-LRHQEGCEWDPWTCEHMFDW-LE (C) (SEQ ID NO: 1602) Con4-35M-Fc-GGGGGAQ-VPRQKDCEWDPWTCEHMYVG-LE (C) (SEQ ID NO: 1602) Con4-43M-Fc-GGGGGAQ-SISHEECEWDPWTCEHMQVG-LE (C) (SEQ ID NO: 1603) Con4-49M-Fc-GGGGGAQ-WAAQEECEWDPWTCEHMGRM-LE (C) (SEQ ID NO: 1604) Con4-27M-Fc-GGGGGAQ-TWPQDKCEWDPWTCEHMGST-LE (C) (SEQ ID NO: 1605) Con4-48M-Fc-GGGGGAQ-GHSQEECGWDPWTCEHMGTS-LE (C) (SEQ ID NO: 1606) Con4-46M-Fc-GGGGGAQ-QHWQEECEWDPWTCDHMPSK-LE (C) (SEQ ID NO: 1607) Con4-41M-Fc-GGGGGAQ-NVRQEKCEWDPWTCEHMPVR-LE (C) (SEQ ID NO: 1608) Con4-36M-Fc-GGGGGAQ-KSGQVECNWDPWTCEHMPRN-LE (C) (SEQ ID NO: 1609) Con4-34M-Fc-GGGGGAQ-VKTQEHCDWDPWTCEHMREW-LE (C) (SEQ ID NO: 1610) Con4-28M-Fc-GGGGGAQ-AWGQEGCDWDPWTCEHMLPM-LE (C) (SEQ ID NO: 1611) Con4-39M-Fc-GGGGGAQ-PVNQEDCEWDPWTCEHMPPM-LE (C) (SEQ ID NO: 1612) Con4-25M-Fc-GGGGGAQ-RAPQEDCEWDPWTCAHMDIK-LE (C) (SEQ ID NO: 1613) Con4-50M-Fc-GGGGGAQ-HGQNMECEWDPWTCEHMFRY-LE (C) (SEQ ID NO: 1614) Con4-38M-Fc-GGGGGAQ-PRLQEECVWDPWTCEHMPLR-LE (C) (SEQ ID NO: 1615) Con4-29M-Fc-GGGGGAQ-RTTQEKCEWDPWTCEHMESQ-LE (C) (SEQ ID NO: 1616) Con4-47M-Fc-GGGGGAQ-QTSQEDCVWDPWTCDHMVSS-LE (C) (SEQ ID NO: 1617) Con4-20M-Fc-GGGGGAQ-QVIGRPCEWDPWTCEHLEGL-LE (C) (SEQ ID NO: 1618) Con4-45M-Fc-GGGGGAQ-WAQQEECAWDPWTCDHMVGL-LE (C) (SEQ ID NO: 1619) Con4-37M-Fc-GGGGGAQ-LPGQEDCEWDPWTCEHMVRS-LE (C) (SEQ ID NO: 1620) Con4-33M-Fc-GGGGGAQ-PMNQVECDWDPWTCEHMPRS-LE (C) (SEQ ID NO: 1621) AC2-Con4M-Fc-GGGGGAQ-FGWSHGCEWDPWTCEHMGST-LE (C) (SEQ ID NO: 1622) Con4-32M-Fc-GGGGGAQ-KSTQDDCDWDPWTCEHMVGP-LE (C) (SEQ ID NO: 1623) Con4-17M-Fc-GGGGGAQ-GPRISTCQWDPWTCEHMDQL-LE (C) (SEQ ID NO: 1624) Con4-8M-Fc-GGGGGAQ-STIGDMCEWDPWTCAHMQVD-LE (C) (SEQ ID NO: 1625) AC4-Con4M-Fc-GGGGGAQ-VLGGQGCEWDPWTCRLLQGW-LE (C) (SEQ ID NO: 1626) Con4-1M-Fc-GGGGGAQ-VLGGQGCQWDPWTCSHLEDG-LE (C) (SEQ ID NO: 1627) Con4-C1M-Fc-GGGGGAQ-TTIGSMCEWDPWTCAHMQGG-LE (C) (SEQ ID NO: 1628) Con4-21M-Fc-GGGGGAQ-TKGKSVCQWDPWTCSHMQSG-LE (C) (SEQ ID NO: 1629) Con4-C2M-Fc-GGGGGAQ-TTIGSMCQWDPWTCAHMQGG-LE (C) (SEQ ID NO: 1630) Con4-18M-Fc-GGGGGAQ-WVNEVVCEWDPWTCNHWDTP-LE (C) (SEQ ID NO: 1631) Con4-19M-Fc-GGGGGAQ-VVQVGMCQWDPWTCKHMRLQ-LE (C) (SEQ ID NO: 1632) Con4-16M-Fc-GGGGGAQ-AVGSQTCEWDPWTCAHLVEV-LE (C) (SEQ ID NO: 1633) Con4-11M-Fc-GGGGGAQ-QGMKMFCEWDPWTCAHIVYR-LE (C) (SEQ ID NO: 1634) Con4-C4M-Fc-GGGGGAQ-TTIGSMCQWDPWTCEHMQGG-LE (C) (SEQ ID NO: 1635) Con4-23M-Fc-GGGGGAQ-TSQRVGCEWDPWTCQHLTYT-LE (C) (SEQ ID NO: 1636) Con4-15M-Fc-GGGGGAQ-QWSWPPCEWDPWTCQTVWPS-LE (C) (SEQ ID NO: 1637) Con4-9M-Fc-GGGGGAQ-GTSPSFCQWDPWTCSHMVQG-LE (C) (SEQ ID NO: 1638) Con4-10M-Fc-GGGGGAQ-TQGLHQCEWDPWTCKVLWPS-LE (C) (SEQ ID NO: 1639) Con4-22M-Fc-GGGGGAQ-VWRSQVCQWDPWTCNLGGDW-LE (C) (SEQ ID NO: 1640) Con4-3M-Fc-GGGGGAQ-DKILEECQWDPWTCQFFYGA-LE (C) (SEQ ID NO: 1641) Con4-5M-Fc-GGGGGAQ-ATFARQCQWDPWTCALGGNW-LE (C) (SEQ ID NO: 1642) Con4-30M-Fc-GGGGGAQ-GPAQEECEWDPWTCEPLPLM-LE (C) (SEQ ID NO: 1643) Con4-26M-Fc-GGGGGAQ-RPEDMCSQWDPWTWHLQGYC-LE (C) (SEQ ID NO: 1644) Con4-7M-Fc-GGGGGAQ-LWQLAVCQWDPQTCDHMGAL-LE (C) (SEQ ID NO: 1645) Con4-12M-Fc-GGGGGAQ-TQLVSLCEWDPWTCRLLDGW-LE (C) (SEQ ID NO: 1646) Con4-13M-Fc-GGGGGAQ-MGGAGRCEWDPWTCQLLQGW-LE (C) (SEQ ID NO: 1647) Con4-14M-Fc-GGGGGAQ-MFLPNECQWDPWTCSNLPEA-LE (C) (SEQ ID NO: 1648) Con4-2M-Fc-GGGGGAQ-FGWSHGCEWDPWTCRLLQGW-LE (C) (SEQ ID NO: 1649) Con4-6M-Fc-GGGGGAQ-WPQTEGCQWDPWTCRLLHGW-LE (C) (SEQ ID NO: 1650) Con4-24M-Fc-GGGGGAQ-PDTRQGCQWDPWTCRLYGMW-LE (C) (SEQ ID NO: 1651) AC1-Con4M-Fc-GGGGGAQ-TWPQDKCEWDPWTCRLLQGW-LE (C) (SEQ ID NO: 1652) AC3-Con4M-Fc-GGGGGAQ-DKILEECEWDPWTCRLLQGW-LE (C) (SEQ ID NO: 1653) AC5-Con4M-Fc-GGGGGAQ-AATQEECEWDPWTCRLLQGW-LE (C) (SEQ ID NO: 1654) L1 DerivedAffinity- Matured Pbs Peptibody Sequence (Seq Id No:) L1-7MGAQ-TNFMPMDDLEQRLYEQFILQQG-LEGGGGG-Fc (N) (SEQ ID NO: 1655) C6-L1MGAQ-TNYKPLDELDATLYEHWILQHS LEGGGGG-Fc (N) (SEQ ID NO: 1656) L1-15MGAQ-QKYQPLDELDKTLYDQFMLQQG LEGGGGG-Fc (N) (SEQ ID NO: 1657) L1-2MGAQ-LNFTPLDELEQTLYEQWTLQQS LEGGGGG-Fc (N) (SEQ ID NO: 1658) L1-10MGAQ-QKFQPLDELEQTLYEQFMLQQA LEGGGGG-Fc (N) (SEQ ID NO: 1659) L1-13MGAQ-QEYEPLDELDETLYNQWMFHQR LEGGGGG-Fc (N) (SEQ ID NO: 1660) L1-5MGAQ-VKYKPLDELDEILYEQQTFQER LEGGGGG-Fc (N) (SEQ ID NO: 1661) L1-C2MGAQ-TKFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (N) (SEQ ID NO: 1662) L1-C3MGAQ-TNFQPLDELDQTLYEQWTLQQR LEGGGGG-Fc (N) (SEQ ID NO: 1663) L1-11MGAQ-QNFKPMDELEDTLYKQFLFQHS LEGGGGG-Fc (N) (SEQ ID NO: 1664) L1-17MGAQ-VKYKPLDELDEWLYHQFTLHHQ LEGGGGG-Fc (N) (SEQ ID NO: 1665) L1-12MGAQ-YKFTPLDDLEQTLYEQWTLQHV LEGGGGG-Fc (N) (SEQ ID NO: 1666) L1-1MGAQ-QNYKPLDELDATLYEHFIFHYT LEGGGGG-Fc (N) (SEQ ID NO: 1667) L1-4MGAQ-VKFKPLDALEQTLYEHWMFQQA LEGGGGG-Fc (N) (SEQ ID NO: 1668) L1-20MGAQ-EDYMPLDALDAQLYEQFILLHG LEGGGGG-Fc (N) (SEQ ID NO: 1669) L1-22MGAQ-YKFNPMDELEQTLYEEFLFQHA LEGGGGG-Fc (N) (SEQ ID NO: 1670) L1-14MGAQ-SNFMPLDELEQTLYEQFMLQHQ LEGGGGG-Fc (N) (SEQ ID NO: 1671) L1-16MGAQ-QKFQPLDELEETLYKQWTLQQR LEGGGGG-Fc (N) (SEQ ID NO: 1672) L1-18MGAQ-QKFMPLDELDEILYEQFMFQQS LEGGGGG-Fc (N) (SEQ ID NO: 1673) L1-3MGAQ-TKFNPLDELEQTLYEQWTLQHQ LEGGGGG-Fc (N) (SEQ ID NO: 1674) L1-21MGAQ-HTFQPLDELEETLYYQWLYDQL LEGGGGG-Fc (N) (SEQ ID NO: 1675) L1-C1MGAQ-QKFKPLDELEQTLYEQWTLQQR LEGGGGG-Fc (N) (SEQ ID NO: 1676) L1-19MGAQ-QTFQPLDDLEEYLYEQWIRRYH LEGGGGG-Fc (N) (SEQ ID NO: 1677) L1-9MGAQ-SKFKLPLDELEQTLYEQWTLQHA LEGGGGG-Fc (N) (SEQ ID NO: 1678) Con1Derived Affinity- Matured Pbs Peptibody Sequence (Seq Id No:) Con1-4M-Fc-GGGGGAQ-SGQLRPCEEIFGCGTQNLAL-LE (C) (SEQ ID NO: 1679) Con1-1M-Fc-GGGGGAQ-AGGMRPYDGMLGWPNYDVQA-LE (C) (SEQ ID NO: 1680) Con1-6M-Fc-GGGGGAQ-GQDLRPCEDMFGCGTKDWYG-LE (C) (SEQ ID NO: 1681) Con1-3M-Fc-GGGGGAQ-APGQRPYDGMLGWPTYQRIV-LE (C) (SEQ ID NO: 1682) Con1-2M-Fc-GGGGGAQ-QTWDDPCMHILGPVTWRRCI-LE (C) (SEQ ID NO: 1683) Con1-5M-Fc-GGGGGAQ-FGDKRPLECMFGGPIQLCPR-LE (C) (SEQ ID NO: 1684) Parent:M-Fc-GGGGGAQ-KRPCEEIFGGCTYQ-LE Con1 (C) (SEQ ID NO: 1685) 12-9 DerivedAffinity- Matured Pbs Peptibody Sequence (Seq Id No:) 12-9-3M-Fc-GGGGGAQ-LQEWCEGVEDPFTFGCEKQR-LE (C) (SEQ ID NO: 1686) 12-9-7M-Fc-GGGGGAQ-MLDYCEGMDDPFTFGCDKQM-LE (C) (SEQ ID NO: 1687) 12-9-6M-Fc-GGGGGAQ-HQEYCEGMEDPFTFGCEYQG-LE (C) (SEQ ID NO: 1688) 12-9-C2M-Fc-GGGGGAQ-LQDYCEGVEDPFTFGCENQR-LE (C) (SEQ ID NO: 1689) 12-9-5M-Fc-GGGGGAQ-LLDYCEGVQDPFTFGCENLD-LE (C) (SEQ ID NO: 1690) 12-9-1M-Fc-GGGGGAQ-GFEYCDGMEDPFTFGCDKQT-LE (C) (SEQ ID NO: 1691) 12-9-4M-Fc-GGGGGAQ-AQDYCEGMEDPFTFGCEMQK-LE (C) (SEQ ID NO: 1692) 12-9-C1M-Fc-GGGGGAQ-LQDYCEGVEDPFTFGCEKQR-LE (C) (SEQ ID NO: 1693) 12-9-2M-Fc-GGGGGAQ-KLEYCDGMEDPFTQGCDNQS-LE (C) (SEQ ID NO: 1694) Parent:M-Fc-GGGGGAQ-FDYCEGVEDPFTFGCDNH-LE 12-9 (C) (SEQ ID NO: 1695)

In addition to the TMP compounds set out in Table 6, the inventionprovides numerous other TMP compounds. In one aspect, TMP compoundscomprise the following general structure:

TMP₁-(L₁)_(n)-TMP₂

wherein TMP₁ and TMP₂ are each independently selected from the group ofcompounds comprising the core structure:

X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀,

wherein,

X₂ is selected from the group consisting of Glu, Asp, Lys, and Val;

X₃ is selected from the group consisting of Gly and Ala;

X₄ is Pro;

X₅ is selected from the group consisting of Thr and Ser;

X₆ is selected from the group consisting of Leu, Ile, Val, Ala, and Phe;

X₇ is selected from the group consisting of Arg and Lys;

X₈ is selected from the group consisting of Gln, Asn, and Glu;

X₉ is selected from the group consisting of Trp, Tyr, and Phe;

X₁₀ is selected from the group consisting of Leu, Ile, Val, Ala, Phe,Met, and Lys;

L₁ is a linker as described herein; and

n is 0 or 1;

and physiologically acceptable salts thereof.

In one embodiment, L₁ comprises (Gly)_(n), wherein n is 1 through 20,and when n is greater than 1, up to half of the Gly residues may besubstituted by another amino acid selected from the remaining 19 naturalamino acids or a stereoisomer thereof.

In addition to the core structure X₂—X₁₀ set forth above for TMP₁ andTMP₂, other related structures are also possible wherein one or more ofthe following is added to the TMP₁ and/or TMP₂ core structure: X₁ isattached to the N-terminus and/or X₁₁, X₁₂, X₁₃, and/or X₁₄ are attachedto the C-terminus, wherein X₁, X₁₂, X₁₃, and X₁₄ are as follows:

X₁ is selected from the group consisting of Ile, Ala, Val, Leu, Ser, andArg;

X₁₁ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Ser, Thr, Lys, His, and Glu;

X₁₂ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Gly, Ser, and Gln;

X₁₃ is selected from the group consisting of Arg, Lys, Thr, Val, Asn,Gln, and Gly; and

X₁₄ is selected from the group consisting of Ala, Ile, Val, Leu, Phe,Thr, Arg, Glu, and Gly.

TMP compounds of the invention are made up of, i.e., comprising, atleast 9 subunits (X₂—X₁₀), wherein X₂—X₁₀ comprise the core structure.The X₂—X₁₄ subunits are amino acids independently selected from amongthe 20 naturally-occurring amino acids, however, the invention embracescompounds where X₂—X₁₄ are independently selected from the group ofatypical, non-naturally occurring amino acids well known in the art.Specific amino acids are identified for each position. For example, X₂may be Glu, Asp, Lys, or Val. Both three-letter and single letterabbreviations for amino acids are used herein; in each case, theabbreviations are the standard ones used for the 20 naturally-occurringamino acids or well-known variations thereof. These amino acids may haveeither L or D stereochemistry (except for Gly, which is neither L norD), and the TMPs (as well as all other compounds of the invention) maycomprise a combination of stereochemistries. The invention also providesreverse TMP molecules (as well as for all other peptides disclosedherein) wherein the amino terminal to carboxy terminal sequence of theamino acids is reversed. For example, the reverse of a molecule havingthe normal sequence X₁₁X₂-X₃ would be X₃—X₂—X₁. The invention alsoprovides retro-reverse TMP molecules (as well as for all other moleculesof the invention described herein) wherein, like a reverse TMP, theamino terminal to carboxy terminal sequence of amino acids is reversedand residues that are normally “L” enantiomers in TMP are altered to the“D” stereoisomer form.

Exemplary TMP compounds of the invention therefore include withoutlimitation the following compounds:

IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (SEQ. ID NO: 993)

(SEQ. ID NO: 994) IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear) (SEQ.ID NO: 995) IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (SEQ. ID NO: 996)IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 997)IEGPTLRQWLAARA-GGGK(BrAc)GGGG-IEGPTLRQWLAARA (SEQ. ID NO: 998)IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 999)IEGPTLRQWLAARA-GGGK(PEG)GGGG-IEGPTLRQWLAARA (SEQ. ID NO: 1000)IEGPTLRQWLAARA-GGGC(PEG)GGGG-IEGPTLRQWLAARA (SEQ. ID NO: 1001)IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (SEQ. ID NO: 1002)

(SEQ. ID NO: 1003) IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ. ID NO:1004) Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (SEQ. ID NO: 1005)Fc-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA-Fc (SEQ. ID NO: 1006)IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA-Fc (SEQ. ID NO: 1007)Fc-GG-IEGPTLRQWLAARA-GPNG-IEGPTLRQWLAARA (SEQ. ID NO: 1008)Fc-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 1009)

(SEQ. IDNO: 1010) Fc-IEGPTLRQCLAARA-GGGGGGGG-IEGPTLRQCLAARA (linear)(SEQ. ID NO: 1011) Fc-IEGPTLRQALAARA-GGGGGGGG-IEGPTLRQALAARA (SEQ. IDNO: 1012) Fc-IEGPTLRQWLAARA-GGGKGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 1013)Fc-IEGPTLRQWLAARA-GGGCGGGG-IEGPTLRQWLAARA (SEQ. ID NO: 1014)Fc-IEGPTLRQWLAARA-GGGNGSGG-IEGPTLRQWLAARA (SEQ. ID NO: 1015)

(SEQ. ID NO: 1016) Fc-GGGGG-IEGPTLRQWLAARA-GGGGGGGG-IEGPTLRQWLAARA (SEQ.ID NO: 1017)

Derivatives

The invention also contemplates derivatizing the peptide and/or vehicleportion (as discussed below) of the compounds. Such derivatives mayimprove the solubility, absorption, biological half life, and the likeof the compounds. The moieties may alternatively eliminate or attenuateany undesirable side-effect of the compounds and the like. Exemplaryderivatives include compounds in which:

1. The compound or some portion thereof is cyclic. For example, thepeptide portion may be modified to contain two or more Cys residues(e.g., in the linker), which could cyclize by disulfide bond formation.For citations to references on preparation of cyclized derivatives, seeTable 2.

2. The compound is cross-linked or is rendered capable of cross-linkingbetween molecules. For example, the peptide portion may be modified tocontain one Cys residue and thereby be able to form an intermoleculardisulfide bond with a like molecule. The compound may also becross-linked through its C-terminus, as in the molecule shown below.

3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by anon-peptidyl linkage. Exemplary non-peptidyl linkages are —CH2-carbamate[—CH2-OC(O)NR—], phosphonate, —CH2-sulfonamide [—CH2-S(O)₂NR-], urea[—NHC(O)NH—], —CH2-secondary amine, and alkylated peptide [—C(O)NR6-wherein R6 is lower alkyl].

4. The N-terminus is derivatized. Typically, the N-terminus may beacylated or modified to a substituted amine. Exemplary N-terminalderivative groups include —NRR1 (other than —NH2), —NRC(O)R1,—NRC(O)OR1, —NRS(O)₂R1, —NHC(O)NHR1, succinimide, orbenzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R1 are each independentlyhydrogen or lower alkyl and wherein the phenyl ring may be substitutedwith 1 to 3 substituents selected from the group consisting of C1-C4alkyl, C1-C4 alkoxy, chloro, and bromo.

5. The free C-terminus is derivatized. Typically, the C-terminus isesterified or amidated. For example, one may use methods described inthe art to add (NH—CH2-CH2-NH2)₂ to compounds of this invention.Likewise, one may use methods described in the art to add —NH2 tocompounds of this invention. Exemplary C-terminal derivative groupsinclude, for example, —C(O)R2 wherein R2 is lower alkoxy or —NR3R4wherein R3 and R4 are independently hydrogen or C1-C8 alkyl (preferablyC1-C4 alkyl).

6. A disulfide bond is replaced with another, preferably more stable,cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al.(1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am.Pep. Symp., 357-9.

7. One or more individual amino acid residues is modified. Variousderivatizing agents are known to react specifically with selectedsidechains or terminal residues, as described in detail below.

8. Heterobifunctional polymers are typically used to link proteins. Anexample is SMCC, orSuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate. The NHS(N-Hyroxylsuccinimide) end reacts with primary amines, which uponconjugation at pH ˜7 is optimal. Once the complex is formed, reaction ofthe maleimide portion of SMCC can proceed with another protein/peptidecontaining a free sulfhydryl group, which occurs at a much faster ratethan the formation of the amide in the initial reaction. The result is alink between two proteins, for example, antibody-enzyme conjugates. Anapplication is illustrated by the preparation of crosslinked Fab′fragments to horseradish peroxidase (Ishikwa, et. al., 1983a,b;Yoshitake et al., 1982a,b; Imagawa et al, 1982; Uto et al., 1991). Theuse of Sulfo SMCC(Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate)allows for water solubility so that an organic solubilization step isnot needed, allowing for greater flexibility and less disruption ofactivity in reacting with proteins.

Lysinyl residues and amino terminal residues may be reacted withsuccinic or other carboxylic acid anhydrides, which reverse the chargeof the lysinyl residues. Other suitable reagents for derivatizingalpha-amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; andtransaminase-catalyzed reaction with glyoxylate.

Arginyl residues may be modified by reaction with any one or combinationof several conventional reagents, including phenylglyoxal,2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization ofarginyl residues requires that the reaction be performed in alkalineconditions because of the high pKa of the guanidine functional group.Furthermore, these reagents may react with the groups of lysine as wellas the arginine epsilon-amino group.

Specific modification of tyrosyl residues has been studied extensively,with particular interest in introducing spectral labels into tyrosylresidues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizole andtetranitromethane are used to form O-acetyl tyrosyl species and 3-nitroderivatives, respectively.

Carboxyl sidechain groups (aspartyl or glutamyl) may be selectivelymodified by reaction with carbodiimides (R′-N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Cysteinyl residues can be replaced by amino acid residues or othermoieties either to eliminate disulfide bonding or, conversely, tostabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med.Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular vehicles. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in Cys, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains.Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman &Co., San Francisco), pp. 79-86 (1983).

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe preferred host cell, optimized codons are known in the art. Codonsmay be substituted to eliminate restriction sites or to include silentrestriction sites, which may aid in processing of the DNA in theselected host cell. The vehicle, linker and peptide DNA sequences may bemodified to include any of the foregoing sequence changes.

Isotope- and toxin-conjugated derivatives. Another set of usefulderivatives are the above-described molecules conjugated to toxins,tracers, or radioisotopes. Such conjugation is especially useful formolecules comprising peptide sequences that bind to tumor cells orpathogens. Such molecules may be used as therapeutic agents or as an aidto surgery (e.g., radioimmunoguided surgery or RIGS) or as diagnosticagents (e.g., radioimmunodiagnostics or RID).

As therapeutic agents, these conjugated derivatives possess a number ofadvantages. They facilitate use of toxins and radioisotopes that wouldbe toxic if administered without the specific binding provided by thepeptide sequence. They also can reduce the side-effects that attend theuse of radiation and chemotherapy by facilitating lower effective dosesof the conjugation partner.

Useful conjugation partners include:

-   -   radioisotopes, such as 90Yttrium, 131Iodine, 225Actinium, and        213Bismuth;    -   ricin A toxin, microbially derived toxins such as Pseudomonas        endotoxin (e.g., PE38, PE40), and the like;    -   partner molecules in capture systems (see below);    -   biotin, streptavidin (useful as either partner molecules in        capture systems- or as tracers, especially for diagnostic use);        and    -   cytotoxic agents (e.g., doxorubicin).

One useful adaptation of these conjugated derivatives is use in acapture system. In such a system, the molecule of the present inventionwould comprise a benign capture molecule. This capture molecule would beable to specifically bind to a separate effector molecule comprising,for example, a toxin or radioisotope. Both the vehicle-conjugatedmolecule and the effector molecule would be administered to the patient.In such a system, the effector molecule would have a short half-lifeexcept when bound to the vehicle-conjugated capture molecule, thusminimizing any toxic side-effects. The vehicle-conjugated molecule wouldhave a relatively long half-life but would be benign and non-toxic. Thespecific binding portions of both molecules can be part of a knownspecific binding pair (e.g., biotin, streptavidin) or can result frompeptide generation methods such as those described herein.

Such conjugated derivatives may be prepared by methods known in the art.In the case of protein effector molecules (e.g., Pseudomonas endotoxin),such molecules can be expressed as fusion proteins from correlative DNAconstructs. Radioisotope conjugated derivatives may be prepared, forexample, as described for the BEXA antibody (Coulter). Derivativescomprising cytotoxic agents or microbial toxins may be prepared, forexample, as described for the BR96 antibody (Bristol-Myers Squibb).Molecules employed in capture systems may be prepared, for example, asdescribed by the patents, patent applications, and publications fromNeoRx. Molecules employed for RIGS and RID may be prepared, for example,by the patents, patent applications, and publications from NeoProbe.

Vehicles

The invention requires the presence of at least one vehicle attached toa peptide through the N-terminus, C-terminus or a sidechain of one ofthe amino acid residues. Multiple vehicles may also be used. In oneaspect, an Fc domain is the vehicle. The Fc domain may be fused to the Nor C termini of the peptides or at both the N and C termini.

In various embodiments of the invention, the Fc component is either anative Fc or an Fc variant. The immunoglobulin source of the native Fcis, in one aspect, of human origin and may, in alternative embodiments,be of any class of immunoglobulin. Native Fc domains are made up ofmonomeric polypeptides that may be linked into dimeric or multimericforms by covalent (i.e., disulfide bonds) and/or non-covalentassociation. The number of intermolecular disulfide bonds betweenmonomeric subunits of native Fc molecules ranges from one to fourdepending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2,IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bondeddimer resulting from papain digestion of an IgG (see Ellison et al.(1982), Nucleic Acids Res. 10: 4071-9).

It should be noted that Fc monomers will spontaneously dimerize when theappropriate cysteine residues are present, unless particular conditionsare present that prevent dimerization through disulfide bond formation.Even if the cysteine residues that normally form disulfide bonds in theFc dimer are removed or replaced by other residues, the monomeric chainswill generally form a dimer through non-covalent interactions. The term“Fc” herein is used to mean any of these forms: the native monomer, thenative dimer (disulfide bond linked), modified dimers (disulfide and/ornon-covalently linked), and modified monomers (i.e., derivatives).

As noted, Fc variants are suitable vehicles within the scope of thisinvention. A native Fc may be extensively modified to form an Fcvariant, provided binding to the salvage receptor is maintained; see,for example WO 97/34631 and WO 96/32478. In such Fc variants, one mayremove one or more sites of a native Fc that provide structural featuresor functional activity not required by the fusion molecules of thisinvention. One may remove these sites by, for example, substituting ordeleting residues, inserting residues into the site, or truncatingportions containing the site. The inserted or substituted residues mayalso be altered amino acids, such as peptidomimetics or D-amino acids.Fc variants may be desirable for a number of reasons, several of whichare described herein. Exemplary Fc variants include molecules andsequences in which:

1. Sites involved in disulfide bond formation are removed. Such removalmay avoid reaction with other cysteine-containing proteins present inthe host cell used to produce the molecules of the invention. For thispurpose, the cysteine-containing segment at the N-terminus may betruncated or cysteine residues may be deleted or substituted with otheramino acids (e.g., alanyl, seryl). Even when cysteine residues areremoved, the single chain Fc domains can still form a dimeric Fc domainthat is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selectedhost cell. For example, one may remove the PA sequence near theN-terminus of a typical native Fc, which may be recognized by adigestive enzyme in E. coli such as proline iminopeptidase. One may alsoadd an N-terminal methionine residue, especially when the molecule isexpressed recombinantly in a bacterial cell such as E. coli.

3. A portion of the N-terminus of a native Fc is removed to preventN-terminal heterogeneity when expressed in a selected host cell. Forthis purpose, one may delete any of the first 20 amino acid residues atthe N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

4. One or more glycosylation sites are removed. Residues that aretypically glycosylated (e.g., asparagine) may confer cytolytic response.Such residues may be deleted or substituted with unglycosylated residues(e.g., alanine).

5. Sites involved in interaction with complement, such as the C1qbinding site, are removed. For example, one may delete or substitute theEKK sequence of human IgG1. Complement recruitment may not beadvantageous for the molecules of this invention and so may be avoidedwith such an Fc variant.

6. Sites are removed that affect binding to Fc receptors other than asalvage receptor. A native Fc may have sites for interaction withcertain white blood cells that are not required for the fusion moleculesof the present invention and so may be removed.

7. The ADCC site is removed. ADCC sites are known in the art; see, forexample, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sitesin IgG1. These sites, as well, are not required for the fusion moleculesof the present invention and so may be removed.

8. When the native Fc is derived from a non-human antibody, the nativeFc may be humanized. Typically, to humanize a native Fc, one willsubstitute selected residues in the non-human native Fc with residuesthat are normally found in human native Fc. Techniques for antibodyhumanization are well known in the art.

An alternative vehicle would be a protein, polypeptide, peptide,antibody, antibody fragment, or small molecule (e.g., a peptidomimeticcompound) capable of binding to a salvage receptor. For example, onecould use as a vehicle a polypeptide as described in U.S. Pat. No.5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also beselected by phage display for binding to the FcRn salvage receptor. Suchsalvage receptor-binding compounds are also included within the meaningof “vehicle” and are within the scope of this invention. Such vehiclesshould be selected for increased half-life (e.g., by avoiding sequencesrecognized by proteases) and decreased immunogenicity (e.g., by favoringnon-immunogenic sequences, as discovered in antibody humanization).

Variants, analogs or derivatives of the Fc portion may be constructedby, for example, making various substitutions of residues or sequences.

Variant (or analog) polypeptides include insertion variants, wherein oneor more amino acid residues supplement an Fc amino acid sequence.Insertions may be located at either or both termini of the protein, ormay be positioned within internal regions of the Fc amino acid sequence.Insertion variants, with additional residues at either or both termini,can include for example, fusion proteins and proteins including aminoacid tags or labels. For example, the Fc molecule may optionally containan N-terminal Met, especially when the molecule is expressedrecombinantly in a bacterial cell such as E. coli.

In Fc deletion variants, one or more amino acid residues in an Fcpolypeptide are removed. Deletions can be effected at one or bothtermini of the Fc polypeptide, or with removal of one or more residueswithin the Fc amino acid sequence. Deletion variants, therefore, includeall fragments of an Fc polypeptide sequence.

In Fc substitution variants, one or more amino acid residues of an Fcpolypeptide are removed and replaced with alternative residues. In oneaspect, the substitutions are conservative in nature and conservativesubstitutions of this type are well known in the art. Alternatively, theinvention embraces substitutions that are also non-conservative.Exemplary conservative substitutions are described in Lehninger,[Biochemistry, 2nd Edition; Worth Publishers, Inc. New York (1975), pp.71-77] and set out immediately below.

CONSERVATIVE SUBSTITUTIONS I SIDE CHAIN CHARACTERISTIC AMINO ACIDNon-polar (hydrophobic): A. Aliphatic A L I V P B. Aromatic F W C.Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S T YB. Amides N Q C. Sulfhydryl C D. Borderline G Positively charged (basic)K R H Negatively charged (acidic) D E

Alternative, exemplary conservative substitutions are set outimmediately below.

CONSERVATIVE SUBSTITUTIONS II ORIGINAL RESIDUE EXEMPLARY SUBSTITUTIONAla (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, ArgAsp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys,Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys(K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro(P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, SerVal (V) Ile, Leu, Met, Phe, Ala

For example, cysteine residues can be deleted or replaced with otheramino acids to prevent formation of some or all disulfide crosslinks ofthe Fc sequences. Each cysteine residue can be removed and/orsubstituted with other amino acids, such as Ala or Ser. As anotherexample, modifications may also be made to introduce amino acidsubstitutions to (1) ablate the Fc receptor binding site; (2) ablate thecomplement (C1q) binding site; and/or to (3) ablate the antibodydependent cell-mediated cytotoxicity (ADCC) site. Such sites are knownin the art, and any known substitutions are within the scope of Fc asused herein. For example, see Molecular Immunology, Vol. 29, No. 5,633-639 (1992) with regard to ADCC sites in IgG1.

Likewise, one or more tyrosine residues can be replaced by phenylalanineresidues. In addition, other variant amino acid insertions, deletionsand/or substitutions are also contemplated and are within the scope ofthe present invention. Conservative amino acid substitutions willgenerally be preferred. Furthermore, alterations may be in the form ofaltered amino acids, such as peptidomimetics or D-amino acids.

Fc sequences of the compound may also be derivatized as described hereinfor peptides, i.e., bearing modifications other than insertion,deletion, or substitution of amino acid residues. Preferably, themodifications are covalent in nature, and include for example, chemicalbonding with polymers, lipids, other organic, and inorganic moieties.Derivatives of the invention may be prepared to increase circulatinghalf-life, or may be designed to improve targeting capacity for thepolypeptide to desired cells, tissues, or organs.

It is also possible to use the salvage receptor binding domain of theintact Fc molecule as the Fc part of a compound of the invention, suchas described in WO 96/32478, entitled “Altered Polypeptides withIncreased Half-Life.” Additional members of the class of moleculesdesignated as Fc herein are those that are described in WO 97/34631,entitled “Immunoglobulin-Like Domains with Increased Half-Lives.” Bothof the published PCT applications cited in this paragraph are herebyincorporated by reference.

WSP Components

Compounds of the invention may further include at least one WSP. The WPSmoiety of the molecule may be branched or unbranched. For therapeuticuse of the end-product preparation, the polymer is pharmaceuticallyacceptable. In general, a desired polymer is selected based on suchconsiderations as whether the polymer conjugate will be usedtherapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. In various aspects,the average molecular weight of each water soluble polymer is betweenabout 2 kDa and about 100 kDa, between about 5 kDa and about 50 kDa,between about 12 kDa and about 40 kDa and between about 20 kDa and about35 kDa. In yet another aspect the molecular weight of each polymer isbetween about 6 kDa and about 25 kDa. The term “about” as used hereinand throughout, indicates that in preparations of a water solublepolymer, some molecules will weigh more, some less, than the statedmolecular weight. Generally, the higher the molecular weight or the morebranches, the higher the polymer/protein ratio. Other sizes may be used,depending on the desired therapeutic profile including for example, theduration of sustained release; the effects, if any, on biologicalactivity; the ease in handling; the degree or lack of antigenicity andother known effects of a water soluble polymer on a therapeutic protein.

The WSP should be attached to a polypeptide or peptide withconsideration given to effects on functional or antigenic domains of thepolypeptide or peptide. In general, chemical derivatization may beperformed under any suitable condition used to react a protein with anactivated polymer molecule. Activating groups which can be used to linkthe water soluble polymer to one or more proteins include withoutlimitation sulfone, maleimide, sulfhydryl, thiol, triflate, tresylate,azidirine, oxirane and 5-pyridyl. If attached to the peptide byreductive alkylation, the polymer selected should have a single reactivealdehyde so that the degree of polymerization is controlled.

Suitable, clinically acceptable, water soluble polymers include withoutlimitation, PEG, polyethylene glycol propionaldehyde, copolymers ofethylene glycol/propylene glycol, monomethoxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly (.beta.-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols(POG) (e.g., glycerol) and other polyoxyethylated polyols,polyoxyethylated sorbitol, or polyoxyethylated glucose, colonic acids orother carbohydrate polymers, Ficoll or dextran and mixtures thereof.

Water Soluble Polymers can also be made to be thermally sensitive, as inthe formation of reverse thermal gels. Examples include Tetronics, withtetra armed backbones, and PEG-PLGA copolymers. The hydrophilicity ofpolymers can be varied by substituting hydrophobic portions into thepolymer chain. An example of this is in the manufacture of PLGA, inwhich the ratio of lactic acid to glycolic acid can be increased toallow for lower water solubility. Lower water soluble polymers may bedesired in certain applications, for example in increasing the potentialto interact with cell membranes. Upon reconstitution, an appropriateratio of phospholipids may be used to induce the formation of micellesor liposomes in solution. The advantage of such a system may be in theability to incorporate some of the protein within the micelle, with thepotential benefit of prolonging delivery. Phospholipids capable offorming liposomes or micelles include DMPG, DMPC, DOPC, DOPG andappropriate secondary liposome strengthening components such ascholesterol. Certain excipients, such as DEA oleth-10 phosphate andoleth 10-phosphate, are capable of forming micelles in solution.

Polysaccharide polymers are another type of water soluble polymer whichmay be used for protein or peptide modification. Modifying proteins orpeptides by adding polysaccharide(s), e.g., glycosylation, may increasehalf-life, decrease antigenicity, increase stability and decreaseproteolysis. Dextrans are polysaccharide polymers comprised ofindividual subunits of glucose predominantly linked by α1-6 linkages.The dextran itself is available in many molecular weight ranges, and isreadily available in molecular weights from about 1 kD to about 70 kD.Dextran is a suitable water soluble polymer for use in the presentinvention as a vehicle by itself or in combination with another vehicle(e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use ofdextran conjugated to therapeutic or diagnostic immunoglobulins has beenreported; see, for example, European Patent Publication No. 0 315 456,which is hereby incorporated by reference. Dextran of about 1 kD toabout 20 kD is preferred when dextran is used as a vehicle in accordancewith the present invention.

In one embodiment, the WSP is PEG and the invention contemplatespreparations wherein a compound is modified to include any of the formsof PEG that have been used to derivatize other proteins, such as andwithout limitation mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The PEG group may be of any convenientmolecular weight and may be linear or branched. The average molecularweight of PEG contemplated for use in the invention ranges from about 2kDa to about 100 kDa, from about 5 kDa to about 50 kDa, from about 5 kDato about 10 kDa. In another aspect, the PEG moiety has a molecularweight from about 6 kDa to about 25 kDa. PEG groups generally areattached to peptides or proteins via acylation or reductive alkylationthrough a reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the target peptide orprotein (e.g., an aldehyde, amino, or ester group). Using methodsdescribed herein, a mixture of polymer/peptide conjugate molecules canbe prepared, and the advantage provided herein is the ability to selectthe proportion of polymer/peptide conjugate to include in the mixture.Thus, if desired, a mixture of peptides with various numbers of polymermoieties attached (i.e., zero, one or two) can be prepared with apredetermined proportion of polymer/protein conjugate.

A useful strategy for the PEGylation (other methods are discussed inmore detail herein) of synthetic peptides consists of combining, throughforming a conjugate linkage in solution, a peptide and a PEG moiety,each bearing a special functionality that is mutually reactive towardthe other. The peptides can be easily prepared with conventional solidphase synthesis. The peptides are “preactivated” with an appropriatefunctional group at a specific site. The precursors are purified andfully characterized prior to reacting with the PEG moiety. Ligation ofthe peptide with PEG usually takes place in aqueous phase and can beeasily monitored by reverse phase analytical HPLC. The PEGylatedpeptides can be easily purified by preparative HPLC and characterized byanalytical HPLC, amino acid analysis and laser desorption massspectrometry.

Linkers

Any “linker” group is optional, whether positioned between peptides,peptide and vehicle or vehicle and WSP. When present, its chemicalstructure is not critical, since it serves primarily as a spacer. Thelinker is preferably made up of amino acids linked together by peptidebonds. Thus, in preferred embodiments, the linker is made up of from 1to 20 amino acids linked by peptide bonds, wherein the amino acids areselected from the 20 naturally occurring amino acids. Some of theseamino acids may be glycosylated, as is well understood by those in theart. In a more preferred embodiment, the 1 to 20 amino acids areselected from glycine, alanine, proline, asparagine, glutamine, andlysine. Even more preferably, a linker is made up of a majority of aminoacids that are sterically unhindered, such as glycine and alanine. Thus,preferred linkers are polyglycines (particularly (Gly)4, (Gly)5, (Gly)8,poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:

(Gly)3Lys(Gly)4; (SEQ ID NO: 1018) (Gly)3AsnGlySer(Gly)2; (SEQ ID NO:1019) (Gly)3Cys(Gly)4; (SEQ ID NO: 1020) and GlyProAsnGlyGly. (SEQ IDNO: 1021)

To explain the above nomenclature, for example, (Gly)3Lys(Gly)4 meansGly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. Combinations of Gly and Ala are alsopreferred. The linkers shown here are exemplary; linkers within thescope of this invention may be much longer and may include otherresidues.

Non-peptide linkers are also possible. For example, alkyl linkers suchas —NH—(CH2)s—C(O)—, wherein s=2-20 could be used. These alkyl linkersmay further be substituted by any non-sterically hindering group such aslower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2,phenyl, etc. An exemplary non-peptide linker is a PEG linker,

wherein n is such that the linker has a molecular weight of 100 to 5000kD, preferably 100 to 500 kD. The peptide linkers may be altered to formderivatives in the same manner as described above.

Polypeptide and Peptide Production

A peptide having been identified may be made in transformed host cellsusing recombinant DNA techniques. If the vehicle component is apolypeptide, the polypeptide- or peptide-vehicle fusion product may beexpressed as one. To do so, a recombinant DNA molecule encoding thepeptide is first prepared using methods well known in the art. Forinstance, sequences coding for the peptides could be excised from DNAusing suitable restriction enzymes. Alternatively, the DNA moleculecould be synthesized using chemical synthesis techniques, such as thephosphoramidate method. Also, a combination of these techniques could beused. The invention therefore provides polynucleotides encoding acompound of the invention.

The invention also provides vectors encoding compounds of the inventionin an appropriate host. The vector comprises the polynucleotide thatencodes the compound operatively linked to appropriate expressioncontrol sequences. Methods of effecting this operative linking, eitherbefore or after the polynucleotide is inserted into the vector, are wellknown. Expression control sequences include promoters, activators,enhancers, operators, ribosomal binding sites, start signals, stopsignals, cap signals, polyadenylation signals, and other signalsinvolved with the control of transcription or translation.

The resulting vector having the polynucleotide therein is used totransform an appropriate host. This transformation may be performedusing methods well known in the art.

Any of a large number of available and well-known host cells may be usedin the practice of this invention. The selection of a particular host isdependent upon a number of factors recognized by the art. These include,for example, compatibility with the chosen expression vector, toxicityof the peptides encoded by the DNA molecule, rate of transformation,ease of recovery of the peptides, expression characteristics, bio-safetyand costs. A balance of these factors must be struck with theunderstanding that not all hosts may be equally effective for theexpression of a particular DNA sequence. Within these generalguidelines, useful microbial hosts include bacteria (such as E. coli),yeast (such as Saccharomyces) and other fungi, insects, plants,mammalian (including human) cells in culture, or other hosts known inthe art.

Next, the transformed host is cultured and purified. Host cells may becultured under conventional fermentation conditions so that the desiredcompounds are expressed. Such fermentation conditions are well known inthe art. Finally, the peptides are purified from culture by methods wellknown in the art.

Depending on the host cell utilized to express a compound of theinvention, carbohydrate (oligosaccharide) groups may conveniently beattached to sites that are known to be glycosylation sites in proteins.Generally, O-linked oligosaccharides are attached to serine (Ser) orthreonine (Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids not countingproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Alternatively, the compounds may be made by synthetic methods. Forexample, solid phase synthesis techniques may be used. Suitabletechniques are well known in the art, and include those described inMerrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis andPanayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis etal. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), SolidPhase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976),The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), TheProteins (3rd ed.) 2: 257-527. Solid phase synthesis is the preferredtechnique of making individual peptides since it is the mostcost-effective method of making small peptides.

Compounds that contain derivatized peptides or which contain non-peptidegroups are particularly amendable to synthesis by well-known organicchemistry techniques.

WSP Modification

For obtaining a compound covalently attached to a WSP, any methoddescribed herein or otherwise known in the art is employed. Methods forpreparing chemical derivatives of polypeptides or peptides willgenerally comprise the steps of (a) reacting the peptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby thepolypeptide becomes attached to one or more polymer molecules, and (b)obtaining the reaction product(s). The optimal reaction conditions willbe determined based on known parameters and the desired result. Forexample, the larger the ratio of polymer molecules:protein, the greaterthe percentage of attached polymer molecule.

A biologically active molecule can be linked to a polymer through anyavailable functional group using standard methods well known in the art.Examples of functional groups on either the polymer or biologicallyactive molecule which can be used to form such linkages include amineand carboxy groups, thiol groups such as in cysteine resides, aldehydesand ketones, and hydroxy groups as can be found in serine, threonine,tyrosine, hydroxyproline and hydroxylysine residues.

The polymer can be activated by coupling a reactive group such astrichloro-s-triazine [Abuchowski, et al., (1977), J. Biol. Chem.252:3582-3586, incorporated herein by reference in its entirety],carbonylimidazole [Beauchamp, et al., (1983), Anal. Biochem. 131:25-33,incorporated herein by reference in its entirety], or succinimidylsuccinate [Abuchowski, et al., (1984), Cancer Biochem. Biophys.7:175-186, incorporated herein by reference in its entirety] in order toreact with an amine functionality on the biologically active molecule.Another coupling method involves formation of a glyoxylyl group on onemolecule and an aminooxy, hydrazide or semicarbazide group on the othermolecule to be conjugated [Fields and Dixon, (1968), Biochem. J.108:883-887; Gaertner, et al., (1992), Bioconjugate Chem. 3:262-268;Geoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146; Gaertner, etal., (1994), J. Biol. Chem. 269:7224-7230, each of which is incorporatedherein by reference in its entirety]. Other methods involve formation ofan active ester at a free alcohol group of the first molecule to beconjugated using chloroformate or disuccinimidylcarbonate, which canthen be conjugated to an amine group on the other molecule to be coupled[Veronese, et al., (1985), Biochem. and Biotech. 11:141-152; Nitecki, etal., U.S. Pat. No. 5,089,261; Nitecki, U.S. Pat. No. 5,281,698, each ofwhich is incorporated herein by reference in its entirety]. Otherreactive groups which may be attached via free alcohol groups are setforth in Wright, EP 0539167A2 (incorporated herein by reference in itsentirety), which also describes the use of imidates for coupling viafree amine groups.

Another chemistry involves acylation of the primary amines of a targetusing the NHS-ester of methoxy-PEG(O—[(N-succinimidyloxycarbonyl)-methyl]-O′-methylpolyethylene glycol).Acylation with methoxy-PEG-NHS results in an amide linkage which willeliminate the charge from the original primary amine. Other methodsutilize mild oxidation of a target under conditions selected to targetthe pendant diol of the penultimate glycosyl unit sialic acid foroxidation to an aldehyde. The resultant glycoaldehyde was then reactedwith a methoxy-PEG-hydrazide(O-(Hydrazinocarbonylmethyl)-O′-methylpolyethylene glycol) to form asemi-stable hydrazone between PEG and target. The hydrazone issubsequently reduced by sodium cyanoborohydride to produce a stable PEGconjugate. See for example, U.S. Pat. No. 6,586,398 (Kinstler, et al.,Jul. 1, 2003), incorporated herein by reference in its entirety.

In specific applications of techniques for chemical modification, forexample, U.S. Pat. No. 4,002,531 (incorporated herein by reference inits entirety) states that reductive alkylation was used for attachmentof polyethylene glycol molecules to an enzyme. U.S. Pat. No. 4,179,337(incorporated herein by reference in its entirety) discloses PEG:proteinconjugates involving, for example, enzymes and insulin. U.S. Pat. No.4,904,584 (incorporated herein by reference in its entirety) disclosesthe modification of the number of lysine residues in proteins for theattachment of polyethylene glycol molecules via reactive amine groups.U.S. Pat. No. 5,834,594 (incorporated herein by reference in itsentirety) discloses substantially non-immunogenic water solublePEG:protein conjugates, involving for example, the proteins IL-2,interferon alpha, and IL-1ra. The methods of Hakimi et al. involve theutilization of unique linkers to connect the various free amino groupsin the protein to PEG. U.S. Pat. Nos. 5,824,784 and 5,985,265 (each ofwhich is incorporated herein by reference in its entirety) teach methodsallowing for selectively N-terminally chemically modified proteins andanalogs thereof, including G-CSF and consensus interferon. Importantly,these modified proteins have advantages as relates to protein stability,as well as providing for processing advantages.

WSP modification is also described in Francis et al., In: Stability ofprotein pharmaceuticals: in vivo pathways of degradation and strategiesfor protein stabilization (Eds. Ahem., T. and Manning, M. C.) Plenum,N.Y., 1991 (incorporated herein by reference in its entirety), is used.In still another aspect, the method described in Delgado et al.,“Coupling of PEG to Protein By Activation With Tresyl Chloride,Applications In Immunoaffinity Cell Preparation”, In: Fisher et al.,eds., Separations Using Aqueous Phase Systems, Applications In CellBiology and Biotechnology, Plenum Press, N.Y., N.Y., 1989 pp. 211-213(incorporated herein by reference in its entirety), which involves theuse of tresyl chloride, which results in no linkage group between theWSP moiety and the polypeptide moiety. In other aspects, attachment of aWSP is effected through use of N-hydroxy succinimidyl esters ofcarboxymethyl methoxy polyethylene glycol, as well known in the art.

For other descriptions of modification of a target with a WSP, see, forexample, U.S. patent application No. 20030096400; EP 0 442724A2; EP0154316; EP 0401384; WO 94/13322; U.S. Pat. Nos. 5,362,852; 5,089,261;5,281,698; 6,423,685; 6,635,646; 6,433,135; International application WO90/07938; Gaertner and Offord, (1996), Bioconjugate Chem. 7:38-44;Greenwald et al., Crit. Rev Therap Drug Carrier Syst. 2000; 17:101-161;Kopecek et al., J Controlled Release., 74:147-158, 2001; Harris et al.,Clin Pharmacokinet. 2001; 40(7):539-51; Zalipsky et al., Bioconjug Chem.1997; 8:111-118; Nathan et al., Macromolecules. 1992; 25:4476-4484;Nathan et al., Bioconj Chem. 1993; 4:54-62; and Francis et al., Focus onGrowth Factors, 3:4-10 (1992), the disclosures of which are incorporatedherein by reference in their entirety.

Reductive Alkylation

In one aspect, covalent attachment of a WSP is carried out by reductivealkylation chemical modification procedures as provided herein toselectively modify the N-terminal α-amino group, and testing theresultant product for the desired biological characteristic, such as thebiological activity assays provided herein.

Reductive alkylation for attachment of a WSP to a protein or peptideexploits differential reactivity of different types of primary aminogroups (e.g., lysine versus the N-terminal) available for derivatizationin a particular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved.

For reductive alkylation, the polymer(s) selected could have a singlereactive aldehyde group. A reactive aldehyde is, for example,polyethylene glycol propionaldehyde, which is water stable, or monoC₁-C₁₀ alkoxy or aryloxy derivatives thereof (see U.S. Pat. No.5,252,714, incorporated herein by reference in its entirety). In oneapproach, reductive alkylation is employed to conjugate a PEG-aldehyde(O-(3-Oxopropyl)-O′-methylpolyethylene glycol) to a primary amine. Underappropriate conditions, this approach has been demonstrated to yield PEGconjugates predominately modified through the α-amine at the proteinN-terminus.

An aldehyde functionality useful for conjugating the biologically activemolecule can be generated from a functionality having adjacent amino andalcohol groups. In a polypeptide, for example, an N-terminal serine,threonine or hydroxylysine can be used to generate an aldehydefunctionality via oxidative cleavage under mild conditions usingperiodate. These residues, or their equivalents, can be normallypresent, for example at the N-terminus of a polypeptide, may be exposedvia chemical or enzymatic digestion, or may be introduced viarecombinant or chemical methods. The reaction conditions for generatingthe aldehyde typically involve addition of a molar excess of sodium metaperiodate and under mild conditions to avoid oxidation at otherpositions in the protein. The pH is preferably about 7.0. A typicalreaction involves the addition of a 1.5 fold molar excess of sodium metaperiodate, followed by incubation for 10 minutes at room temperature inthe dark.

The aldehyde functional group can be coupled to an activated polymercontaining a hydrazide or semicarbazide functionality to form ahydrazone or semicarbazone linkage. Hydrazide-containing polymers arecommercially available, and can be synthesized, if necessary, usingstandard techniques. PEG hydrazides for use in the invention can beobtained from Shearwater Polymers, Inc., 2307 Spring Branch Road,Huntsville, Ala. 35801 (now part of Nektar Therapeutics, 150 IndustrialRoad, San Carlos, Calif. 94070-6256). The aldehyde is coupled to thepolymer by mixing the solution of the two components together andheating to about 37° C. until the reaction is substantially complete. Anexcess of the polymer hydrazide is typically used to increase the amountof conjugate obtained. A typical reaction time is 26 hours. Depending onthe thermal stability of the reactants, the reaction temperature andtime can be altered to provide suitable results. Detailed determinationof reaction conditions for both oxidation and coupling is set forth inGeoghegan and Stroh, (1992), Bioconjugate Chem. 3:138-146, and inGeoghegan, U.S. Pat. No. 5,362,852, each of which is incorporated hereinby reference in its entirety.

Using reductive alkylation, the reducing agent should be stable inaqueous solution and preferably be able to reduce only the Schiff baseformed in the initial process of reductive alkylation. Reducing agentsare selected from, and without limitation, sodium borohydride, sodiumcyanoborohydride, dimethylamine borate, trimethylamine borate andpyridine borate.

The reaction pH affects the ratio of polymer to protein to be used. Ingeneral, if the reaction pH is lower than the pK_(a) of a targetreactive group, a larger excess of polymer to protein will be desired.If the pH is higher than the target pK_(a), the polymer:protein rationeed not be as large (i.e., more reactive groups are available, so fewerpolymer molecules are needed).

Accordingly, the reaction is performed in one aspect at a pH whichallows one to take advantage of the pK_(a) differences between theε-amino groups of the lysine residues and that of the α-amino group ofthe N-terminal residue of the protein. By such selective derivatization,attachment of a water soluble polymer to a protein is controlled; theconjugation with the polymer takes place predominantly at the N-terminusof the protein and no significant modification of other reactive groups,such as the lysine side chain amino groups, occurs.

In one aspect, therefore, methods are provided for covalent attachmentof a WSP to a target compound and which provide a substantiallyhomogenous preparation of WSP/protein conjugate molecules, in theabsence of further extensive purification as is required using otherchemical modification chemistries. More specifically, if polyethyleneglycol is used, methods described allow for production of anN-terminally PEGylated protein lacking possibly antigenic linkagegroups, i.e., the polyethylene glycol moiety is directly coupled to theprotein moiety without potentially toxic by-products.

Depending on the method of WSP attachment chosen, the proportion of WSPmolecules attached to the target peptide or protein molecule will vary,as will their concentrations in the reaction mixture. In general, theoptimum ratio (in terms of efficiency of reaction in that there is noexcess unreacted protein or polymer) is determined by the molecularweight of the WSP selected. In addition, when using methods that involvenon-specific attachment and later purification of a desired species, theratio may depend on the number of reactive groups (typically aminogroups) available.

Purification

The method of obtaining a substantially homogeneous WSP-modifiedpreparation is, in one aspect, by purification of a predominantly singlespecies of modified compound from a mixture of species. By way ofexample, a substantially homogeneous species is first separated by ionexchange chromatography to obtain material having a chargecharacteristic of a single species (even though other species having thesame apparent charge may be present), and then the desired species isseparated using size exclusion chromatography. Other methods arereported and contemplated by the invention, includes for example, PCT WO90/04606, published May 3, 1990, which describes a process forfractionating a mixture of PEG-protein adducts comprising partitioningthe PEG/protein adducts in a PEG-containing aqueous biphasic system.

Thus, one aspect of the present invention is a method for preparing aWSP-modified compound conjugate comprised of (a) reacting a compoundhaving more than one amino group with a water soluble polymer moietyunder reducing alkylation conditions, at a pH suitable to selectivelyactivate the α-amino group at the amino terminus of the protein moietyso that said water soluble polymer selectively attaches to said α-aminogroup; and (b) obtaining the reaction product. Optionally, andparticularly for a therapeutic product, the reaction products areseparated from unreacted moieties.

As ascertained by peptide mapping and N-terminal sequencing, apreparation is provided which comprises at least 50% PEGylated peptidein a mixture of PEGylated peptide and unreacted peptide. In otherembodiments, preparations are provided which comprises at least 75%PEGylated peptide in a mixture of PEGylated peptide and unreactedpeptide; at least 85% PEGylated peptide in a mixture of PEGylatedpeptide and unreacted peptide; at least 90% PEGylated peptide in amixture of PEGylated peptide and unreacted peptide; at least 95%PEGylated peptide in a mixture of PEGylated peptide and unreactedpeptide; and at least 99% PEGylated peptide in a mixture of PEGylatedpeptide and unreacted peptide.

The following examples are not intended to be limiting but onlyexemplary of specific embodiments of the invention.

Example 1 mFc-TMP Expression Construct Assembly

A polynucleotide encoding a TMP fusion protein comprising a murine Fcregion (mFc-TMP) was constructed by combining nucleotide sequencesindividually encoding murine Fc and a TMP (described in EP01124961A2).In the first round of PCR, the murine Fc-encoding component wasamplified with PCR primers 3155-58 (SEQ ID NO: 1022) and 1388-00 (SEQ IDNO: 1023).

3155-58: CCGGGTAAAGGTGGAGGTGGTGGTATCGA (SEQ ID NO: 1024) 3155-59:CCACCTCCACCTTTACCCGGAGAGTGGGAG (SEQ ID NO: 1025)

In a separate reaction, a TMP-encoding polynucleotide was amplified withprimers 1209-85 (SEQ ID NO: 1026) and 3155-59 (SEQ ID NO: 1027).

1209-85: CGTACAGGTTTACGCAAGAAAATGG (SEQ ID NO: 1028) 1388-00:CTAGTTATTGCTCAGCGG (SEQ ID NO: 1029)

The resulting PCR fragments were gel purified and combined in a singletube for a second round of PCR with primers 1209-85 (SEQ ID NO: 1030)and 1388-00 (SEQ ID NO: 1031). The PCR product from this second round ofamplification was gel purified and digested with restriction enzymesNdeI and XhoI. The digestion fragment was purified and ligated into thevector pAMG21, previously digested with the same enzymes. This ligationmix was transformed via electroporation into E. coli and plated ontoLB+Kanamycin media. Colonies were screened via PCR and DNA sequencing. Apositive clone with a nucleotide sequence (SEQ ID NO: 1032) encoding themFc-TMP fusion protein (SEQ ID NO: 1033) was identified and designated6397.

Murine Fc-TMP fusion protein-encoding polynucleotide (SEQ ID NO: 1034)

  1 GATTTGATTC TAGATTTGTT TTAACTAATT AAAGGAGGAA TAACAT Open RF:ATGGTCGACGGTTG TAAGCCATGC ATTTGTACAG TCCCAGAAGT ATCATCTGTC 101TTCATCTTCC CCCCAAAGCC CAAGGATGTG CTCACCATTA CTCTGACTCC 151 TAAGGTCACGTGTGTTGTGG TAGACATCAG CAAGGATGAT CCCGAGGTCC 201 AGTTCAGCTG GTTTGTAGATGATGTGGAGG TGCACACAGC TCAGACGCAA 251 CCCCGGGAGG AGCAGTTCAA CAGCACTTTCCGCTCAGTCA GTGAACTTCC 301 CATCATGCAC CAGGACTGGC TCAATGGCAA GGAGTTCAAATGCAGGGTCA 351 ACAGTGCAGC TTTCCCTGCC CCCATCGAGA AAACCATCTC CAAAACCAAA401 GGCAGACCGA AGGCTCCACA GGTGTACACC ATTCCACCTC CCAAGGAGCA 451GATGGCCAAG GATAAAGTCA GTCTGACCTG CATGATAACA GACTTCTTCC 501 CTGAAGACATTACTGTGGAG TGGCAGTGGA ATGGGCAGCC AGCGGAGAAC 551 TACAAGAACA CTCAGCCCATCATGGACACA GATGGCTCTT ACTTCGTCTA 601 CAGCAAGCTC AATGTGCAGA AGAGCAACTGGGAGGCAGGA AATACTTTCA 651 CCTGCTCTGT GTTACATGAG GGCCTGCACA ACCACCATACTGAGAAGAGC 701 CTCTCCCACT CTCCGGGTAA AGGTGGAGGT GGTGGTATCG AAGGTCCGAC751 TCTGCGTCAG TGGCTGGCTG CTCGTGCTGG TGGTGGAGGT GGCGGCGGAG 801GTATTGAGGG CCCAACCCTT CGCCAATGGC TTGCAGCACG CGCATAA 3′ Sequence:TCTCGAGGATCCG CGGAAAGAAG AAGAAGAAGA AGAAAGCCCG AAAGG

Murine Fc-TMP protein sequence (SEQ ID NO: 1035)

  1 MVDGCKPCIC TVPEVSSVFI FPPKPKDVLT ITLTPKVTCV VVDISKDDPE  51VQFSWFVDDV EVHTAQTQPR EEQFNSTFRS VSELPIMHQD WLNGKEFKCR 101 VNSAAFPAPIEKTISKTKGR PKAPQVYTIP PPKEQMAKDK VSLTCMITDF 151 FPEDITVEWQ WNGQPAENYKNTQPIMDTDG SYFVYSKLNV QKSNWEAGNT 201 FTCSVLHEGL HNHHTEKSLS HSPGKGGGGGIEGPTLRQWL AARAGGGGGG 251 GGIEGPTLRQ WLAARA*

Example 2 Fermentation of Strain 6397

Fermentation of strain 6397 was initiated by inoculation of 500 mL ofsterilized Luria broth with a seed culture of the strain in a shakeflask. When cell density reached 0.9 at 600 nm, the contents were usedto inoculate a 15 L fermentor containing 10 L of complex based growthmedium (800 g glycerol, 500 g trypticase, 3 g sodium citrate, 40 gKH₂PO₄, 20 g (NH₄)₂SO₄, 5 ml Fluka P-2000 antifoam, 10 ml trace metals(ferric chloride 27.0 g/L, zinc chloride 2.00 g/L, cobalt chloride 2.00g/L, sodium molybdate 2.00 g/L, calcium chloride 1.00 g/L, cupricsulfate 1.90 g/L, boric acid 0.50 g/L, manganese chloride 1.60 g/L,sodium citrate dihydrate 73.5 g/L), 10 ml vitamins (biotin 0.060 g/L,folic acid 0.040 g/L, riboflavin 0.42 g/L, pyridoxine HCl 1.40 g/L,niacin 6.10 g/L, pantothenic acid 5.40 g/L, sodium hydroxide 5.30 ml/L),add water to bring to 10 L). The fermenter was maintained at 37° C. andpH 7 with dissolved oxygen levels kept at a minimum of 30% saturation.When the cell density reached 13.1 OD units at 600 nm, the culture wasinduced by the addition 10 ml of 0.5 mg/ml N-(3-oxo-hexanoyl) homoserinelactone. At 6 hours post induction, the broth was chilled to 10° C., andthe cells were harvested by centrifugation at 4550 g for 60 min at 5° C.The cell paste was then stored at −80° C.

Example 3 Protein Refolding

E. coli paste (300 g) from strain 6397 expressing mFc-TMP was dissolvedin 2250 ml lysis buffer (50 mM Tris HCl, 5 mM EDTA, pH 8.0) and passedthrough a chilled microfluidizer two times at 13,000 PSI. The homogenatewas then centrifuged at 11,300 g for 60 minutes at 4° C. The supernatantwas discarded, and the pellet was resuspended in 2400 ml of water usinga tissue grinder. The homogenate was then centrifuged at 11,300 g for 60minutes at 4° C. The supernatant was discarded, and the pellet wasresuspended in 200 ml volumes of water using a tissue grinder. Thehomogenate was centrifuged at 27,200 g for 30 minutes at 4° C., and thesupernatant was discarded. About 12.5% of the pellet was resuspended in28 ml 20 mM Tris HCl, pH 8.0, with 35 mg hen egg white lysozyme (Sigma,St Louis, Mo.) using a tissue grinder and incubated at 37° C. for 20min. Following incubation, the suspension was centrifuged at 27,200 gfor 30 minutes at 22° C., and the supernatant was discarded. The pelletwas resuspended in 35 ml 8 M guanidine HCl, 50 mM Tris HCl, pH 8.0,after which 350 μl 1 M DTT (Sigma, St Louis, Mo.) was added and materialwas incubated at 37° C. for 30 minutes. The solution was thencentrifuged at 27,200 g for 30 minutes at 22° C. The supernatant wasthen transferred to 3.5 L of refolding buffer (50 mM Tris base, 160 mMarginine HCl, 3 M urea, 20% glycerol, pH 9.5, 1 mM cysteine, 1 mMcystamine HCl) at 1 ml/min with gentle stirring at 4° C.

Example 4 Construct Purification

After about 40 hours incubation at 4° C. with gentle agitation, therefold solution described in Example 3 was concentrated to 500 μl usinga tangential flow ultrafiltration apparatus with a 30 kDa cartridge(Satorius, Goettingen, Germany) followed by diafiltration against 3 L ofQ-Buffer A (20 mM Tris HCl, pH 8.0). The concentrated material wasfiltered through a Whatman GF/A filter and loaded on to an 86 mlQ-Sepharose fast flow column (2.6 cm ID) (Amersham Biosciences,Piscataway, N.J.) at 15 ml/min. After washing the resin with severalcolumn volumes of Q-Buffer A, the protein was eluted using a 20 columnvolume linear gradient to 60% Q-Buffer B (20 mM Tris HCl, 1 M NaCl, pH8.0) at 10 ml/min. The peak fractions were pooled, and the pool waspassed through a Mustang E syringe filter (Pall Corporation, East Hills,N.Y.) at 1 ml/min. The filtered material was filtered a second timethrough a 0.22 μm cellulose acetate filter and stored at −80° C.

Example 5 Protein PEGylation

To a cooled (4° C.), stirred solution of mFc-TMP (3.5 ml, 0.8 mg/ml) ina 100 mM sodium acetate buffer, pH 5, containing 20 mM NaCNBH₃, wasadded a 3.8-fold molar excess of methoxypolyethylene glycol aldehyde(MPEG) (average molecular weight, 20 kDa) (Nektar). The stirring of thereaction mixture was continued at the same temperature. The extent ofthe protein modification during the course of the reaction was monitoredby SEC HPLC using a Superose 6 HR 10/30 column (Amersham Biosciences)eluted with a 0.05 M phosphate buffer with 0.15 M NaCl, pH 7.0 at 0.4ml/min. After 16 hours the SEC HPLC analysis indicated that the majorityof the protein has been conjugated to MPEG. At this time the reactionmixture was buffer-exchanged into a 20 mM Tris/HCl buffer, pH 8.12. TheMPEG-mFc-AMP2 conjugates were isolated by ion exchange chromatographyusing a 1 ml Hi Trap HP Q column (Amersham Biosciences) equilibratedwith a 20 mM Tris/HCl buffer, pH 8.12. The reaction mixture was loadedon the column at a flow rate of 0.5 ml/min and the unreacted MPEGaldehyde was eluted with three column volumes of the starting buffer. Alinear 20-column-volume gradient from 0% to 100% 20 mM Tris/HCl buffer,pH 8.12, containing 0.5 M NaCl was used to the elute the protein-polymerconjugates. Fractions (2 ml) collected during ion exchangechromatography separation were analyzed by HPLC SEC as described above.A fraction containing the mono- and di-MPEG-mFc-TMP conjugates in anapproximate ratio of 2.3 to 1 (as determined by SEC HPLC) wasconcentrated, and sterile filtered.

Example 6 In vivo Testing

BDF1 mice (Charles River Laboratories, Wilmington, Mass.) were dividedinto groups of 10 and injected on days 0, 21, and 42 subcutaneously witheither diluting agent (Dulbecco's PBS with 0.1% bovine serum albumin) ordiluting agent with 50 μg test mono- and di-MPEG-mFc-TMP conjugateprotein (as described above) per kg animal. Each group was divided inhalf and bled (140 μl) from the retro-orbital sinus on alternate timepoints (days 0, 3, 5, 7, 10, 12, 14, 19, 24, 26, 28, 31, 33, 40, 45, 47,49, 52 and 59). On day 59, mice were anesthetized with isoflurane priorto bleeding. The collected blood was analyzed for a complete anddifferential count using an ADVIA 120 automated blood analyzer withmurine software (Bayer Diagnostics, New York, N.Y.).

Example 7 Lyophilized Human Fc-TMP

Initial Lyophilized Formulation Screening Studies

The human Fc-TMP peptibody described herein in Example 7 corresponds toa dimeric form of SEQ ID NO:1017, wherein the human Fc is SEQ ID NO:1,having an initiator methionine at the N-terminus.

The stability of Fc-TMP was assessed by several chromatographictechniques: Reversed-Phase HPLC, Cation-Exchange HPLC, Size-ExclusionHPLC and SDS-PAGE, all of which were stability-indicating at elevatedtemperature. Formulations ranging in concentration from 0.1 to 40 mg/mlwere examined for both chemical and physical degradation at accelerated,refrigerated and frozen temperature. Fc-TMP stability was evaluated withrespect to varying pH and the inclusion of mannitol or glycine ascake-forming agents and sucrose as a lyoprotectant. Mannitol and sucrosewere eventually chosen for further optimization after the othercandidate (glycine) showed no improvement in protein stability. Tween-20(polysorbate-20) was also shown to inhibit aggregation uponlyophilization over a concentration range of 0.002 to 0.1%. Thefollowing buffers were examined in screening studies over a pH range of4-8: glycine, succinate, histidine, phosphate, and Tris. From thesescreening studies, Fc-TMP formulated in histidine buffer at pH 5 with asmall amount of Tween-20 (0.004%) added was shown to be more optimal forstability.

Validation of Sucrose and Tween-20 in the Fc-TMP Formulation

Subsequent development efforts were focused on validating the level ofsucrose, mannitol and Tween-20 in the formulation at a proteinconcentration of approximately 0.5 mg/ml (to accommodate the anticipateddosing requirements in the clinic). The effect of sucrose, mannitol andTween-20 in optimizing stability was demonstrated in these studies.Follow-up studies were also initiated for the purpose of anticipatingmanufacturing issues and concerns.

Sucrose is Beneficial in Minimizing Chemical Degradation at ElevatedTemperature

The effect of varying sucrose and mannitol concentrations on thestability of Fc-TMP was tested. The protein was formulated at 0.3 and 2mg/ml in order to bracket the anticipated concentration range in theclinic. In addition, samples were prepared with and without 0.004%Tween-20. The ratio of sucrose:mannitol was changed by varying theamount of sucrose and adjusting the mannitol level at each sucroseconcentration to maintain isotonicity. The following ratios ofsucrose:mannitol were examined (expressed as percent weight per volume):0.2:5.1; 0.5:4.8; 1:4.5; 1.5:4.3 and 2:4.

Higher ratios of sucrose:mannitol are shown to minimize chemicaldegradation as monitored by Cation-Exchange and Reversed-Phase HPLC. Asis shown in Table 39, the percent main peak is compared initially andafter elevated temperature storage of Fc-TMP for 18 weeks at 37° C. Thegreatest loss of cation-exchange main peak occurs in the liquidformulation (Fc-TMP formulated in 10 mM acetate, 5% sorbitol at pH 5),followed by the lyophilized formulations with 0.2, 0.5 and 1.0% sucrose,respectively. The protective effect of sucrose in minimizing chemicaldegradation was also observed by Reversed-Phase HPLC analysis of samplesafter elevated temperature storage (Table 39). The percent main peak(determined from reversed-phase HPLC analysis of Fc-TMP) dropssignificantly at the low sucrose levels, but does not appear to changemeaningfully in formulations with sucrose concentrations of greater than1%. Interpreted together, these results indicate that maintainingsucrose levels at 1.5% or higher is critical for the stability of Fc-TMPupon lyophilization.

TABLE 39 Fc-TMP in 10 mM Histidine, buffered at pH 5 with Tween-20 Lossof RP and CEX-HPLC Main Peak After 18 Weeks at 37° C. RP-HPLC CEX-HPLCFormulation Time Zero 18 Weeks Time Zero 18 Weeks 0.2% Sucrose, 78.672.2 79.8 62.6 5.1% Mannitol 0.5% Sucrose, 77.3 73.1 78.9 71.6 4.8%Mannitol 1% Sucrose, 78.4 78.0 80.5 73.9 4.5% Mannitol 1.5% Sucrose,73.2 79.8 80.5 78.7 4.3% Mannitol 2% Sucrose, 79.2 81.3 78.6 78.9 4%Mannitol 10 mM Acetate, 74.7 42.8 75.5 34.1 5% Sorbitol, pH 5 (liquidcontrol)

Whereas Fc-TMP in the liquid control (the 10 mM acetate, 5% sorbitol, pH5 formulation) has significant growth in the pre- and post-main peakarea, the protein shows more degradation in the post-main peak regionupon analysis of lyophilized samples with lower amounts of sucrose.Previous work with liquid stability samples (after elevated temperaturestorage) has shown that deamidation arises from glutamine and argininein the protein, which contributes to the growth in the pre-peak regionin liquid samples.

At refrigerated temperature, chemical degradation is not observed bycation-exchange and reversed-phase HPLC in the lyophilized formulationafter long term storage. For example, cation-exchange chromatograms didnot show apparent changes under varied temperatures (−80° C., 4° C. andcontrolled room temperature for 6 months). Due to the lack of chemicaldegradation in the lyophilized formulation over time at controlled roomtemperature and lower, much of the formulation development work centeredon minimizing the physical aggregation associated with freeze-drying.

Tween-20 Minimizes Aggregation Induced by Lyophilization

The inclusion of Tween-20 at a low concentration (0.004%) is needed tominimize a small amount of aggregation which is apparent followinglyophilization. This can be demonstrated by examining the relevantresults from several stability studies in which samples are evaluatedfor stability with and without the addition of Tween-20.

A higher protein concentration of Fc-TMP was first used to explore awide range of Tween-20 in order to investigate the amount needed tominimize aggregation. The Fc-TMP concentration in this study was 20mg/ml, with the Tween-20 levels set at 0.002, 0.004, 0.006 and 0.01%.After storage for one year at 4° C., aggregation is limited to <0.1% inall formulations with Tween-20. Six month results also showed nomeaningful aggregation. Tween-20 at 0.004% was chosen for furtherconsideration in the formulation studies, as discussed herein, designedfor Fc-TMP at 0.5 mg/ml.

Table 40 shows the amount of aggregation in Fc-TMP monitored at timezero, 3 and 11 months after storage at 4° C. In this study, Fc-TMP waslyophilized at 0.5 mg/ml in the aforementioned formulation and informulations with varying sucrose:mannitol ratios without Tween-20added. In addition, stability was followed in the current formulationwithout Tween-20 and buffered at pH 4.5, 5 and 5.5. Results show thatonly the aforementioned formulation has minimal aggregation at pH 5. Informulations without Tween-20, aggregation varies from 0.5% to around5%. Aggregation is also higher at pH 4.5 and 5.5 compared to thatdetected at pH 5. Lower sucrose:mannitol ratios (0.2, 0.5 and 1% sucroseformulations) have higher aggregation, as levels are typically around5%. Over time, the level of aggregation remains consistent in theaforementioned formulation and in the formulation without Tween-20through the 1 year timepoint.

TABLE 40 Fc-TMP in 10 mM Histidine, varied formulations, 0.5 mg/mlSEC-HPLC Measured Percent Aggregation Time Formulation Zero 3 Months 11Months¹ 2% Sucrose, 4% Mannitol, pH 5.0 <0.1 <0.1 <0.1 (with 0.004%Tween-20) 2% Sucrose, 4% Mannitol, pH 4.5 2.4 2.9 — 2% Sucrose, 4%Mannitol, pH 5.0 0.5 1.6   0.8 2% Sucrose, 4% Mannitol, pH 5.5 2.3 2.5 —0.2% Sucrose, 5.1% Mannitol, pH 5.0 4.8 7.3 — 0.5% Sucrose, 4.8%Mannitol, pH 5.0 5.1 4.4 — 1% Sucrose, 4.5% Mannitol, pH 5.0 4.5 4.3 —¹The optimized formulation samples were selected for evaluation at the11 month timepoint.

Additional formulation studies were designed to confirm the beneficialeffect of Tween-20 in minimizing aggregation. All of the samples inthese studies were formulated at pH 5 with and without 0.004% Tween-20.Table 41 lists the percent aggregation after storage at 4° C. for timeintervals of zero, 18 weeks and 1 year in stability. At time zero,immediately following lyophilization, aggregation is minimized in all ofthe samples with 0.004% Tween-20. Small amounts of aggregation areobserved in samples without Tween-20, with the highest amount found inthe formulation with 0.2% sucrose. The effectiveness of Tween-20 inminimizing aggregation also extends to the 18 week timepoint, withhigher percent aggregation found in samples lacking Tween-20 and at lowsucrose: mannitol ratios. After storage for one year at 4° C.,aggregation is also consistently low in the samples containing Tween-20.

TABLE 41 Fc-TMP in 10 mM Histidine, varied formulations, 0.3 mg/ml, pH 5SEC-HPLC Measured Percent Aggregation after Storage at 4° C. FormulationTime Zero 4 Months 1 Year¹ 2% Sucrose, 4% Mannitol <0.1 0.2 <0.1 (with0.004% Tween-20) 2% Sucrose, 4% Mannitol 0.2 0.2 — 0.2% Sucrose, 5.1%Mannitol <0.1 <0.1 <0.1 (with 0.004% Tween-20) 0.2% Sucrose, 5.1%Mannitol 1.1 1.3 — 0.5% Sucrose, 4.8% Mannitol <0.1 0.2 <0.1 (with0.004% Tween-20) 0.5% Sucrose, 4.8% Mannitol 0.2 0.8 — 1% Sucrose, 4.5%Mannitol <0.1 <0.1 <0.1 (with 0.004% Tween-20) 1% Sucrose, 4.5% Mannitol0.1 0.3 — 1.5% Sucrose, 4.8% Mannitol <0.1 <0.1 <0.1 (with 0.004%Tween-20) 1.5% Sucrose, 4.8% Mannitol 0.1 0.3 — ¹Samples with Tween-20were selected for evaluation at the 1 year timepoint.

Another stability study, designed to test the effectiveness ofantioxidants in minimizing chemical degradation, reinforced theprotective effect of Tween-20. Antioxidants did not have an impact inminimizing chemical degradation upon elevated temperature storage.However, the aforementioned formulation, with 0.004% Tween-20 and at anFc-TMP concentration of 0.2 mg/ml, had <0.1% aggregation at time zeroand 0.1% after 5 months storage at 4° C. The same formulation withoutTween-20 had 0.4% aggregation at time zero and 1% aggregation afterstorage for 5 months.

The stability study results presented in Tables II, III and theaforementioned studies illustrate the protective effect of Tween-20 inminimizing aggregation upon lyophilization. The growth of aggregationover time is minimal, as timepoints extending to 1 year at 4° C. show nomeaningful increases in aggregation in samples formulated with 0.004%Tween-20. Based on these stability study results which show that theaddition of Tween-20 minimizes aggregation upon lyophilization, scale-upwork was initiated with the recommended formulation.

Scale-Up Studies

Aggregation is Concentration Dependent

An initial scale-up study was designed to simulate manufacturingconditions and examine the robustness of the formulation with respect toshipping stress and stability upon reconstitution. Fc-TMP was bufferexchanged into the formulation buffer using a tangential flow filtrationdevice, similar to larger scale processes. The protein was subsequentlydiluted to concentrations of 0.5 and 0.1 mg/ml with Tween-20 also addedprior to the final filtration step. After samples were filled,lyophilization was performed. An exemplary lyophilzation process is setforth below:

Thermal Treatment Steps Temp Time Ramp/Hold Step #1 −50 120 R Step #2−50 120 H Step #3 −13 60 R Step #4 −13 360 H Step #5 −50 60 R Step #6−50 60 H Freeze Temp −50 C. Additional Freeze 0 min Condenser Setpoint−60 C. Vacuum Setpoint 100 mTorr Primary Drying Steps temp time VacRamp/Hold Step #1 −50 15 100 H Step #2 −25 120 100 R Step #3 −25 600 100H Step #4 −25 600 100 H Step #5 0 800 100 R Step #6 25 800 100 R Step #725 800 100 H Step #8 25 800 100 H Step #9 25 0 100 H Step #10 25 0 100 HStep #11 25 0 100 H Step #12 25 0 100 H Step #13 25 0 100 H Step #14 250 100 H Step #15 25 0 100 H Step #16 25 0 100 H Post Heat 25 100 100 HSecondary Temperature 28 C.

Passive storage at 4° C. resulted in more aggregation at the low Fc-TMPconcentration (0.1 mg/ml). At time zero, aggregation was determined bySEC-HPLC to be 0.4% in the 0.1 mg/ml formulation, whereas 0.1%aggregation was detected in the protein formulated at 0.5 mg/ml. Aftersix months storage at refrigerated temperature, the aggregation remainedat the same levels as observed for the time zero samples for bothconcentrations of Fc-TMP, in agreement with results from previousstability studies. Due to the higher amount of aggregation observed atthe lowest concentration (0.1 mg/ml) it was decided that 0.5 mg/ml wouldbe best suited as the concentration of choice for additional scale-upwork.

Aggregation Does Not Increase Upon Simulated Shear Stress

Lyophilized samples (not reconstituted) from the initial scale-up studywere also subjected to simulated ground and air transportation with theaid of stress simulation equipment. Briefly, the protocol as outlined inthe ASTM (American Society of Testing Methods), Method # D-4728, wasfollowed. Simulated ground and air transportation was accomplished usingan Electrodynamic Vibration Table, Model S202, and a Power Amplifier,Model # TA240 (Unholtz-Dickie Corporation, Wallingford, Conn.).Following the transportation stress, the physical appearance of thelyophilized cakes was compared to passive controls, with the result thatno morphological changes of the cake were obvious. Both chemical andphysical stability was acceptable, with aggregation consistent in thestressed samples and passive controls (<0.1% in the 0.5 mg/ml samplescompared to 0.4% in the 0.1 mg/ml samples).

Stability upon reconstitution was examined in this study by preparingfreshly reconstituted samples and incubating for 3, 7 and 14 days eitherpassively, with slow tumbling, or vigorous shaking. Table 42 shows theresults for the 0.1 and 0.5 mg/ml formulations. As is expected, theamount of aggregation is minimized in the formulations at 0.5 mg/ml.Compared to the slow tumbling over time vs. the non-tumbled samples, nomeaningful increase in aggregation is apparent over the 14 day period.The amount of dimerization in these formulations (Non-tumbling andTumbling) is also consistent. Interestingly, the shaking results appearto display a trend; i.e. the aggregation drops to less than detectablelevels after time zero in both the 0.1 and 0.5 mg/ml samples. Meanwhile,there is a corresponding increase in the amount of dimerization observedin most shaken samples at each timepoint, suggesting that there is somereversibility in going from the aggregate to the dimer state upon shearstressing Fc-TMP.

TABLE 42 Fc-TMP in 10 mM Histidine, with 2% Sucrose, 4% Mannitol and0.004% Tween-20, pH 5 SEC-HPLC Measured Percent Aggregation andDimerization after Storage at 4° C. Time Zero 3 Days 7 Days 14 Days agg,dimer agg, dimer agg, dimer agg, dimer 0.1 mg/ml Non-Tumbling 0.4, 0.31.2, 0.6 1.1, 0.4 1.0, 0.4 Tumbling 0.4, 0.3 0.7, 0.4 0.7, 0.3 1.1, 0.3Shaking 0.4, 0.3 <0.1, 0.9   <0.1, 0.1   <0.1, 1.6   0.5 mg/mlNon-Tumbling 0.1, 0.5 0.2, 0.5 0.2, 0.5 0.2, 0.6 Tumbling 0.1, 0.5 0.2,0.6 0.1, 0.6 0.1, 0.6 Shaking 0.1, 0.5 <0.1, 0.9   <0.1, 1.0   <0.1,2.2  

Secondary Drying for 12 Hours in the Lyophilization Cycle is Sufficientfor Minimizing Residual Moisture

A second scale-up study was performed in which Fc-TMP wasbuffer-exchanged and diluted to 0.5 mg/ml in the recommendedformulation. Lyophilization was achieved with a cycle consisting of aninitial freezing step at −50° C., followed by annealing at −13° C. Thetemperature was then ramped down to −50° C., held for an hour, andprimary drying initiated at −50° C. with a vacuum setpoint of 100 mTorr.Following a brief hold period at −50 C, the temperature was ramped downto −25° C. over a two hour period, maintained at −25° C. for 20 hours,and then gradually raised to 25° C. after around 27 hours for thebeginning of secondary drying. Secondary drying was continued for aminimum of 12 hours at 25° C. During the lyophilization cycle, sampleswere pulled after 12, 18 and 24 hours of secondary drying in order tocheck the stability and compare the level of residual moisture. Resultsshow that residual moisture, as measured by Karl Fisher Titration, isaround 0.6% or less (Table 43) in all samples examined. Moisture issimilarly low in the buffer placebo cakes. Based on this work, thesecondary drying time of the lyophilization cycle can be shortened to arange between 12-18 hours.

TABLE 43 Residual Moisture in Fc-TMP Secondary Drying Times Held at 12,18 and 24 Hours Secondary Drying Time Karl Fisher Percent Moisture 12hours 0.23 12 hours 0.38 Buffer Placebo 0.43 18 hours 0.63 18 hours 0.28Buffer Placebo 0.3 24 hours 0.46 24 hours 0.37 Buffer Placebo 0.31

Stability results are also comparable over this secondary drying timerange, as samples do not have differences with respect to chemical orphysical stability. For example, the amount of aggregation is <0.1% forall samples examined, while the percent dimer is consistently at 0.1%.These results confirm that the secondary drying time can be shortened toless than 24 hours without affecting the initial stability of theprotein.

Additional work was performed to evaluate the robustness of theformulation with respect to varying excipient concentrations. Sinceprevious stability work had shown that sucrose, for example, can have animpact on stability, it was necessary to examine the robustness ofFc-TMP upon minor changes in excipient levels.

Statistical Study of Fc-TMP Robustness of Formulation

An initial statistical study had been designed to examine minor changesin formulation variables such as the formulation pH, histidine bufferstrength and the sucrose:mannitol ratio, using an E-chip softwarepackage. These samples were lyophilized but a non-optimal freeze-drycycle was used which resulted in higher aggregation than typicallyobserved. The study was a screening study, assuming a linear responsesurface. Results of the stability assessment showed that the pH (4.7 to5.3) and histidine buffer strength, (varied from 5 to 15 mM), had littleimpact on the stability of Fc-TMP. In order to verify the contributionof Tween-20 and the sucrose:mannitol ratio in the overall stability ofFc-TMP, a follow-up statistical design study was initiated using themore optimal lyophilization cycle.

Quadratic Statistical Stability Study

The second statistical design study examined variations in Tween-20(0.001%, 0.0045% and 0.008%), the sucrose:mannitol ratio (1.7:4.2, 2:4and 2.3:3.8), and variations in the protein concentration (0.3, 0.65 and1 mg/ml). The pH of the formulations were also adjusted to 4.7, 5 and5.3, and the Histidine buffer strength varied from 7, 10 and 13 mM.Samples were prepared and lyophilized using a Virtis lyophilizer (SPIndustries, Inc., Gardiner, N.Y.), with an optimized conservative cycleused for the previous stability studies. Stability results wereinterpreted using E-chip (statistical design software package,Hockessin, Del.) in two ways: an assessment of the impact of formulationvariables on the amount of aggregation and dimerization observed at timezero, and the effect of formulation variables in affecting elevatedtemperature (37° C.) storage stability as measured by rates of changefrom time zero.

Time Zero Formulation Results from Quadratic Statistical Study

Results from the time zero assessment showed that, as expected, Tween-20minimizes aggregation, however the protein concentration was alsosignificant in reducing the tendency to aggregate upon freeze-drying.The E-chip software program assesses the effects that different inputvariables (the formulation conditions) have on Fc-TMP aggregation anddimerization during freeze-drying. With respect to Fc-TMP dimerizationat time zero, not one excipient of the formulation was considered (basedon E-chip results) to have a significant effect. Several formulationvariables impacted the amount of aggregation observed upon freeze-dryingFc-TMP. Based on the summary results provided by E-chip, the Fc-TMPconcentration had the greatest effect on the degree of aggregationobserved at time zero, followed by the Tween-20 level. Aggregation isobserved to be the highest, at time zero, in the low concentrationsamples. Likewise, higher amounts of Tween-20 have more of a protectiveeffect in minimizing aggregation at time zero, although this trend isnot as meaningful as the protein concentration effect. Higher amounts ofaggregation were observed in this study compared to previous studyresults, and around 0.5% aggregation was observed in some samples at thelower protein concentrations with Tween-20.

It was observed that the variability in aggregation drops as the proteinconcentration increases. At 0.5 mg/ml and higher concentrations, Fc-TMPhas average lower aggregation than in samples formulated at or below 0.3mg/ml.

Tween-20 at 0.004% is Effective in Minimizing Aggregation upon ElevatedTemperature Storage

The Statistical Design study was also used to assess changes uponelevated temperature storage. Rates of aggregation were compared in thevaried formulation conditions after 16 days at 37° C. by subtracting theelevated temperature results from the initial (time zero) results andnormalizing to one month's time. Rates which are negative thus refer toan apparent loss of the measured property over time. Response variables(corresponding to results from assays) were determined through RP-HPLC(percent main-peak purity and pre-peak percent), cation-exchange HPLC(percent main-peak purity), size-exclusion HPLC (aggregation and dimerformation) and NIR-water (residual moisture by infra-red spectroscopy,which was correlated with Karl Fisher Titration results in some samplesto verify accuracy).

Results from a comparison of the rates of change obtained from eachassay technique show that changes in aggregation and RP-HPLC oxidationwere statistically significant with respect to the Fc-TMP concentrationand Tween-20 squared concentration, to within two standard deviations.The Tween-20 squared term most likely arises from the quadratic natureof the study which assumes a curved response surface. In this case, thesquared Tween-20 term fits the model better, in addition to possiblysuggesting that there is an interactive effect with itself which affectsstability. The other measured responses, such as cation-exchange HPLCmain-peak purity, or the varied pH conditions, for example, did notexhibit any significant responses in affecting protein stability.

As is the case with the initial scale-up study previously discussed(tumbled, non-tumbled and shaken samples), the amount of aggregation islower after high temperature storage. The rate of change in thesesamples was used to make predictions based on the statistical model(quadratic study) to anticipate the protective effect of Tween-20. Table44 shows predictions for the amount of aggregation expected, based onthe statistical design model, as the Tween-20 concentration is raisedfrom 0 to 0.008%. As is shown, the rate of aggregation, normalized toone month at 37° C., is negative (indicating the loss of aggregate inthese conditions) with the exception of 0% Tween-20, in which caseaggregation would be predicted to grow. The rate of loss of aggregateappears to plateau at Tween-20 concentrations of 0.002% and higher,suggesting that Tween-20 at low levels (0.002-0.006%) is sufficient inminimizing physical degradation. The rate of growth of dimer iscorrespondingly similar under these conditions and was not statisticallycorrelated with any of the formulation excipients, as previouslymentioned.

TABLE 44 Statistical Quadratic Model Predictions Varied Tween-20 andFc-TMP Concentration Based on 16 Days Incubation at 37° C. (normalizedto one month) % Fc-TMP % SEC % RP-HPLC Tween 20 (mg/ml) Aggregationprediction limits Oxidation prediction limits 0 0.5 0.09 (−0.32, 0.50)  0.47 (−2.59, 1.65) 0.002 0.5 −0.35 (−0.69, −0.0)  −0.68 (−2.44, 1.08)0.004 0.5 −0.53 (−0.87, −0.19) −0.58 (−2.32, 1.17) 0.006 0.5 −0.45(−0.78, −0.12) −0.17 (−1.86, 1.52) 0.008 0.5 −0.12 (−0.46, 0.21)   0.54(−1.18, 2.26) 0.004 0.3 −0.59 (−0.94, −0.25) 0.58 (−1.18, 2.34) 0.0040.1 −0.63 (−1.10, −0.16) 2.69   (0.28, 5.09)

The rate of change in RP-HPLC measured oxidation at 0.5 mg/ml, assumingthat this corresponds to changes in the pre-peak region of eachchromatogram, is not statistically significant with respect to theTween-20 squared term. In this case, the model (as shown in Table 44)predicts that as the Tween-20 concentration is varied the rate ofoxidation is consistent with the limits of the prediction intervals. Theprotein concentration affects oxidation, as the rate of growth increasesfrom 0.58 to 2.69 as the protein concentration drops from 0.3 to 0.1mg/ml (while keeping the Tween-20 constant at 0.004%.).

These results suggest that maintaining the Tween-20 concentrationbetween 0.002 to 0.006% is desirable from a stability standpoint. Theamount of protein is also important, as the stability is worse atconcentrations below 0.5 mg/ml.

Refrigerated Temperature Stability

Considering the above, the following formulation was used for assessingthe refrigerated temperature stability of lyophilized Fc-TMP: 0.5 mg/mlFc-TMP in 10 mM Histidine buffered at pH 5, 2% Sucrose, 4% Mannitol, and0.004% Tween-20.

The formulation was monitored for refrigerated temperature stability fora period of one year. Table 45 shows the stability results from thisstudy, with results from time zero, 3 months and 1 year listed. As isshown, the percent main peak purity, as measured by Reversed-Phase andCation-Exchange HPLC, does not appear to decrease with time. The minordifferences in main-peak purity over time are typical of the normalvariation in resolution of the chromatographic columns and are alsoobserved in the frozen standard. The percent aggregation is consistentand does not appear to grow after one year.

TABLE 45 Timepoint Concentration 0 3 months 1 Year Reversed-Phase HPLCPercent Main Peak 0.3 81.0 82.5 82.4 0.5 69.0 82.7 83.4 1.0 80.6 82.483.2 Frozen Starting Material 82.4 84.0 84.5 Cation-Exchange HPLCPercent Main Peak 0.3 67.0 71.8 81.8 0.5 73.5 83.1 80.0 1.0 74.2 79.780.5 Frozen Starting Material 75.3 77.0 83.6 Size-Exclusion HPLC PercentAggregation 0.3 <0.1 0.1 0.1 0.5 <0.1 <0.1 0.1 1.0 <0.1 <0.1 <0.1 FrozenStarting Material 0.1 0.1 <0.1

Fc-TMP is formulated in 10 mM Histidine, buffered at pH 5.0 with 2%sucrose, 4% mannitol and 0.004% Tween-20. It has been shown that pH 5 ismore optimal for stability, and that the sucrose:mannitol ratio iscritical in minimizing chemical degradation upon storage at elevatedtemperature for this protein system. Tween-20 is needed at a lowconcentration in order to minimize the amount of aggregation whichoccurs as a result of the lyophilization-process. Stability studies forscale-up applications support these conclusions. Statistical studieshave also been designed which validate the level of each excipient inthe formulation, including the need for the protein concentration to beat 0.5 mg/ml. Refrigerated temperature stability of the recommendedformulation does not show meaningful degradation after storage for oneyear at 4° C.

Example 8 Lyophilized Fc-Ang-2 Binding Peptide

In order to determine the optimal formulation for lyophilized Fc-Ang-2binding peptide, analyses were carried out that assessed Fc-Ang-2binding peptide aggregation and stability at various pH values,excipient concentrations, and protein concentrations.

Fc-Ang-2 binding peptide consists of two pharmaceutically activepolypeptide molecules linked to the C-terminus of the Fc portion of anIgG1 antibody molecule. The molecule is comprised of 574 amino acidresidues with a total molecular weight of 63,511 Daltons. The pI of themolecule is 5.45. There are two disulfide bonds on each of the activepolypeptides. There are a total of 20 cysteine residues throughout themolecule, most of which are oxidized in disulfide bridges. The sequenceof Fc-Ang-2 binding peptide is as follows:

(SEQ ID NO: 2) MDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGGAQQEECEWDPWTCEHMGSGSATGGSGSTASSGSGSATHQEECEWDPWTCEHMLE

The Fc portion of the IgG1 terminates at K228. G229-G233 composes alinker sequence. The active polypeptide begins at A234 and extends tothe rest of the sequence.

Lyophilized Fc-Ang-2 Binding Peptide pH Screen

The pH screen tested the stability of an Fc-Ang-2 binding peptide at pH4.0, 7.0, and 8.0. At pH 4.0, the screened buffers included glutamicacid, sodium citrate, and sodium succinate, each at a concentration of10 mM. At pH 7.0, the screened buffers were histidine and tris, both at10 mM concentration. Hisitidine and Tris were also screened at pH 8.0,each at 10 mM. Each of the buffers contained 4% mannitol as alyophilization caking agent and 2% sucrose as an excipient. In addition,the histidine buffer at pH 7.0 was examined with and without thepresence of a surfactant, 0.01% Tween 20 (w/w). The protein was dilutedto 5 mg/ml with each of the formulation buffers. This solution was thendialyzed into each of the formulation buffers using dialysis tubing witha 10,000 Da molecular weight exclusion limit, where a total of 6exchanges were performed with a minimum of 4 hours of equilibrationbetween exchanges. After dialysis, the protein was aliquoted into 3 ccglass vials with a 1 ml fill volume. These vials were then lyophilizedusing a lab-scale freeze-drying instrument. After lyophilization, thevials were sealed and stored for incubation at 4° C., 29° C., and 37°C., where individual vials were pulled and analyzed at various pointsover time, beginning immediately after lyophilization and extending outto a period of 24 months. The samples were reconstituted with theappropriate volume of water and analyzed for protein stability usingsize exclusion liquid chromatography and gel electrophoresis (whichdetect aggregation, dimerization and proteolytic cleavage), anionexchange liquid chromatography (which detects oxidation). In addition,the properties of the cake such as reconstitution times and moisture andproperties of the reconstituted liquid solution were analyzed (such aspH).

Lyophilized Fc-Ang-2 Binding Peptide Excipient Study

The excipient screen was performed in a single buffer, 10 mM hisitidineat pH 7.0. The two excipients compared in this study were 0.85% arginineand 1% sucrose. The caking agent used with arginine was 4% mannitol, andthe caking agent used with sucrose was 2% glycine. Each of theformulations was tested at protein concentrations of 1 mg/ml, 30 mg/ml,and 60 mg/ml. In addition, one formulation containing a manntiol sucrosecombination was tested at 30 mg/ml. Each of the formulations contained0.01% Tween 20. The protein was first concentrated to 70 mg/ml anddialyzed into the appropriate formulation using a lab-scaleultrafiltration/diafiltration device. The protein was then diluted toeach of the three concentrations with the appropriate formulationbuffer. The protein was then aliquoted into 3 cc glass vials with a fillvolume of 1 ml. The vials were then lyophilized using a lab-scalefreeze-drying instrument. After lyophilization, the vials were sealedand stored for incubation at 4° C., 29° C., 37° C., and 52° C., whereindividual vials were pulled and analyzed at various points over time,beginning immediately after lyophilization and extending out to a periodof 24 months. The samples were analyzed for protein stability using sizeexclusion chromatography, anion exchange chromatography and SDS-PAGE.The properties of the lyophilized cake and reconstituted liquidsolutions were also analyzed.

Lyophilized Fc-Ang-2 Binding Peptide Concentration Screen

The concentration screen was performed in 10 mM histidine at pH 7.2,with 4% mannitol as the caking agent and 2% sucrose as an excipient. Theprotein was concentrated to approximately 140 mg/ml and dialyzed intothe formulation using a lab-scale ultrafiltration/diafiltration device.The dialyzed protein was then diluted to 30 mg/ml, 60 mg/ml, and 120mg/ml in the formulation buffer. The solutions were then aliquoted into3 cc glass vials at a fill volume of 1 ml. The vials were lyophilizedusing a lab-scale freeze-drying instrument. After lyophilization, thevials were sealed and stored for incubation at 4° C., 29° C., 37° C.,and 52° C., where individual vials were pulled and analyzed at variouspoints over time, beginning immediately after lyophilization andextending out to a period of 24 months. The samples were analyzed forprotein stability using size exclusion chromatography, anion exchangechromatography and SDS-PAGE. The properties of the lyophilized cake andreconstituted liquid solutions were also analyzed.

Conclusion

Considering the above, the optimal formulation(s) comprises 10 mMhistidine, 4% mannitol, 2% sucrose, 0.01% Tween-20, pH 7.0.

Example 9 Lyophilized Fc-Agp-3 Binding Peptide

In order to determine the optimal formulation for lyophilized Fc-Agp-3binding peptide, analyses were carried out that assessed Fc-Agp-3binding peptide aggregation and stability at various pH values,excipient concentrations, and protein concentrations.

Fc-Agp-3 binding peptide is a N-linked peptibody against the B cellactivation factor (BAFF) aimed against B cell-related diseases. Thepeptibody is constructed of two non-glycosylated disulfide-linkedpolypeptides with a total mass of ˜63.6 kD. The isoelectric point forthis peptibody has been estimated to be pH 7.36.

Fc SEQUENCE (SEQ ID NO: 1696):

V D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S RT P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K PR E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K AL P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R D E L T K N Q VS L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L DS D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N HY T Q K S L S L S P G K

Agp-3 Binding Peptide Sequence (SEQ ID NO: 1697):

G C K W D L L I K Q W V C D P L G S G S A T G G S G S T A S S G S G S AT H M L P G C K W D L L I K Q W V C D P L G G G G G

Thus, the sequence of Fc-Agp-3 binding peptide binding peptide is asfollows:

(SEQ ID NO: 1698) G C K W D L L I K Q W V C D P L G S G S A T G G S G ST A S S G S G S A T H M L P G C K W D L L I K Q W V C D P L G G G G G VD K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S R TP E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K P RE E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K A LP A P I E K T I S K A K G Q P R E P Q V Y T L P P S R D E L T K N Q V SL T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L D SD G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N H YT Q K S L S L S P G K

Lyophilized Fc-Agp-3 Binding Peptide Broad pH Screen at 10 mg/ml

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC),which was stability-indicating at elevated temperatures. To assess thestability and reconstitution properties of lyophilized Fc-Agp-3 bindingpeptide over the pH range of 3.85-7.6, 10 mg/mL Fc-Agp-3 binding peptidewas formulated in various 10 mM buffers in the presence of 2.5% mannitoland 2.0% sucrose. The following buffers were tested at approximately 0.5pH unit increments: acetate, succinate, histidine, pyrophosphate,phosphate and Tris.

For the formulation development work, purified bulk material wasobtained in the 30 mg/mL frozen liquid formulation. The material wasdialyzed into the appropriate formulation buffers and lyophilized in aVirtis lyophilizer using a conservative cycle. The annealing step wasperformed at −20° C. and lasted for 4 hours to allow for mannitolcrystallization. The primary drying was performed at the shelftemperature of −25° C. for 20 hours. The primary drying reachedcompletion at −25° C. since no spike in the vacuum was observed as theshelf temperature was increased up to 0° C. No major collapse wasobserved and the samples proceeded successfully through the secondarydrying first at 0° C., and then at 25° C. Upon reconstitution theformulations with pH at or above 7 were slightly turbid, while all theother formulations were clear. This was explained by the proximity ofthe high pH formulations to the isoelectric point of Fc-Agp-3 bindingpeptide (pI=7.36). SE-HPLC analysis revealed a dimer as the main highmolecular weight specie. It was observed that relative % dimer wasstrongly pH dependent with the lowest accumulation at pH 5 and below.The amount of soluble aggregates also showed some pH dependence. Nosoluble aggregates were observed in the samples prior to lyophilization.In contrast, a small amount of dimer was present in all formulationsprior to lyophilization, and it increased further in the reconstitutedsamples, post lyophilization. No significant clipping was observed inall formulations. The highest amount of the intact monomer was in thecase of acetate, succinate and histidine formulations at pH 5 and below.

Lyophilized Fc-Agp-3 Binding Peptide Broad pH Screen at 30 mg/ml:Stabilizing Effect of Sucrose and Mannitol

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC),which was stability-indicating. To evaluate the effect of the presenceof 2.5% mannitol and 3.5% sucrose on stability and reconstitutionproperties of lyophilized Fc-Agp-3 binding peptide over the pH range of4.5-7.5, Fc-Agp-3 binding peptide was formulated at 30 mg/mL in 10 mMsuccinate, histidine and phosphate buffers. Pyrophosphate and Trisbuffers were excluded from this study due to poor performance in theprevious broad pH screen. Acetate buffer was excluded due to possibilityof pH changes in the reconstituted samples as a result of acetatesublimation during freeze-drying.

For the formulation development work, purified bulk material wasobtained in the 30 mg/mL frozen liquid formulation. The material wasdialyzed into the appropriate formulation buffers and lyophilized in aVirtis lyophilizer using a conservative cycle. The annealing step wasperformed at −20° C. and lasted for 5 hours to allow for more completemannitol crystallization. The primary drying was performed at the shelftemperature of −25° C. for 20 hours. The primary drying not quitereached completion at −25° C., and some spike in the vacuum was observedas the shelf temperature was increased up to 0° C. No major collapse wasobserved and the samples proceeded successfully through the secondarydrying first at 0° C., and then at 25° C. Upon reconstitutionformulations with pH around 7 were slightly turbid, while all otherformulations were clear. As it was mentioned previously, this could beexplained by the proximity of the high pH formulations to theisoelectric point of Fc-Agp-3 binding peptide. SE-HPLC analysis revealeddimer as the main high molecular weight specie. Again, it was observedthat relative % dimer was strongly pH dependent with the lowestaccumulation at pH 5 and below. The amount of soluble aggregates did notshow clear pH dependence. No soluble aggregates were observed in thesamples prior to lyophilization. ˜0.6% dimer was observed in the non-GMPbulk prior to formulating and it increased up to 3.0% for high pHformulations prior to lyophilization as a result of bufferexchange/concentration.

The pH dependence of the dimer accumulation was also confirmed by closecorrespondence of the relative amount dimer at a given pH irrespectiveof the type of buffer. % dimer did not increase significantlypost-lyophilization as evidenced by T=0 samples. The only exception werethe samples non-containing both, sucrose and mannitol, which showednoticeable, ˜0.25-0.5% increase in % dimer. In the presence of sucrosethe main peak loss was minimal even after buffer exchange/lyophilizationfor succinate and histidine formulations with pH 4.1 and 4.7,respectively. Compared to them, all phosphate formulations showed 3%loss of the main peak prior to lyophilization. Although, the cake wasformed even in the absence of both sucrose and mannitol, thecorresponding main peak dropped by 0.5-0.7% compared to thesugar-containing formulations. In addition, reconstitution of thenon-sugar formulations was much longer (>2 min) and required someagitation. In order to ensure robustness of the cake mannitol or glycinewere included as bulking agents in all subsequent formulations eventhough sucrose alone was shown to confer sufficient protein stability.

Lyophilized Fc-Agp-3 Binding Peptide Narrow pH Screen at 30 mg/ml

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC)and reversed-phase HPLC (RP-HPLC), which were stability-indicating atelevated temperatures. Since % recovery of the main peak was higher atpH 5 and below, the phosphate buffer was omitted from the narrow pHscreen. In addition to the succinate and histidine buffers, whichperformed well in the broad pH screens, the narrow pH screen included 10mM glutamate, pH 4-6. The formulations were tested at 0.2 pH unitincrements. The sucrose and mannitol content was kept constant at 3.5%and 2.5%, respectively, except for two succinate formulations at pH 4.5with 2.0% and 5.0% sucrose. Also, six potential generic lyo formulationswere tested at every pH unit increments, such as:

1) 20 mM histidine, 2.0% glycine, 1.0% sucrose at pH 5.0

2) 20 mM histidine, 2.0% glycine, 1.0% sucrose at pH 6.0

3) 20 mM histidine, 2.0% glycine, 1.0% sucrose at pH 7.0

4) 20 mM histidine, 4.0% mannitol, 2.0% sucrose at pH 5.0

5) 20 mM histidine, 4.0% mannitol, 2.0% sucrose at pH 6.0

6) 20 mM histidine, 4.0% mannitol, 2.0% sucrose at pH 7.0

For the formulation development work, purified bulk material wasobtained in the 30 mg/mL frozen liquid formulation. The material wasdialyzed into the appropriate formulation buffers and lyophilized in aVirtis lyophilizer using a conservative cycle. The lyophilization cyclewas further modified. The annealing step was performed at −15° C. toallow for glycine crystallization and lasted for 5 hours. The primarydrying was performed initially at −30° C. for a short period of time (4h). Then the shelf temperature was raised to −25° C. and kept constantfor 24 hours. However, primary drying did not quite reach completion at−25° C. as evidenced by a small spike in the vacuum as the shelftemperature was further increased up to 0° C. Nevertheless, no majorcollapse was observed and the samples proceeded successfully through thesecondary drying first at 0° C., and then at 25° C. Up to 6 monthslyophilized state stability data was generated at 37° C. to assesslong-term stability of Fc-Agp-3 binding peptide. Increase in pH resultsin the loss of the main peak for all histidine formulations. Onlygeneric formulations were monitored up to 6 months, but the advantage ofthe pH near 5.0 was already obvious for the 1- and 3-month timepoints.Moreover, 6-month data for the generic formulations suggests thatmannitol+sucrose formulations are more stable than glycine+sucrose,especially at pH 6 and higher.

In the case of glutamate and succinate formulations there was also aclear pH-dependent increase in the amount of dimer, the main degradationproduct, as pH becomes less acidic. Similar pH dependence is seen forthe aggregates. The highest recovery of the main peak was observed forpH 5 and below. Initial loss of the main peak for T=0 can be explainedby protein degradation during buffer exchange and concentrating asevidenced by a similar loss for the formulations prior tolyophilization. However, 3-month stability data suggest that glutamateformulations had higher physical stability than their succinatecounterparts (with the same pH). It has to be noted that in this studywe also tested effect of increased sucrose concentrations on thestability of succinate formulations. In addition to 3.5% sucrose, wecompared 2.0% and 5.0% sucrose formulations in succinate, pH 4.5. Theincrease in sucrose decreased high molecular weight species to someextent, but it did not significantly affect the amount of the main peak.Therefore, 3.5% sucrose was considered optimal since such formulationsmost closely matched with physiological tonicity.

One of clip specie was observed growing at low pH by RP-HPLC. Asignificant portion of the pH-dependent clipping in the lyophilizedformulations occurred prior to lyophilization as a result oftime-consuming buffer exchange and protein concentrating steps. Afterlyophilization no significant increase in the amount of clips wasobserved even after 3- to 6-month storage at 37° C. In general the datasuggests using higher pH formulations to mitigate the clipping since itis unobservable at pH 6 and above. However, the amount of the dimer issignificant at higher pH and may be as high as 2.5-4.5% at pH 6 andabove. Therefore, a compromise can be found in formulating Fc-Agp-3binding peptide at pH 5, where clipping is moderate, especially in theglutarnate and histidine buffers, and when the dimer formation is stillsufficiently suppressed.

Conclusion

2.5% mannitol and 3.5% sucrose have provided sufficient cake and proteinstability as confirmed by 6-month shelf study at 37° C. Therefore, 10 mMhistidine, 2.5% mannitol, 3.5% sucrose at pH 5.0 and 10 mM glutamate,2.5% mannitol, 3.5% sucrose at pH 5.0 can be used for formulating 30mg/mL Fc-Agp-3 binding peptide. In addition, this study shows that 20 mMhistidine, 4.0% mannitol, 2.0% sucrose at pH 5.0 also performs well andcan be considered as a possible generic lyo formulation for peptibodies.

Example 10 Lyophilized Fc-Myo Binding Peptide

In order to determine the optimal formulation for lyophilized Fc-Myobinding peptide, analyses were carried out that assessed Fc-Myo bindingpeptide aggregation and stability at various pH values, excipientconcentrations, and protein concentrations.

Fc-Myo binding peptide is a C-linked peptibody against the myostatinprotein aimed against muscle wasting-related diseases. The peptibody isconstructed of two non-glycosylated disulfide-linked polypeptides with atotal mass of ˜59.1 kD. The isoelectric point for this peptibody hasbeen estimated to be pH 6.88.

Fc Sequence (SEQ ID NO:1699):

M D K T H T C P P C P A P E L L G G P S V F L F P P K P K D T L M I S RT P E V T C V V V D V S H E D P E V K F N W Y V D G V E V H N A K T K PR E E Q Y N S T Y R V V S V L T V L H Q D W L N G K E Y K C K V S N K AL P A P I E K T I S K A K G Q P R E P Q V Y T L P P S R D E L T K N Q VS L T C L V K G F Y P S D I A V E W E S N G Q P E N N Y K T T P P V L DS D G S F F L Y S K L T V D K S R W Q Q G N V F S C S V M H E A L H N HY T Q K S L S L S P G K

Myostatin Binding Peptide Sequence (SEQ ID NO:1700):

G G G G G A Q L A D H G Q C I R W P W M C P P E G W E

Thus, the sequence of Fc-Myo binding peptide binding peptide is asfollows:

(SEQ ID NO: 1701) M D K T H T C P P C P A P E L L G G P S V F L F P P KP K D T L M I S R T P E V T C V V V D V S H E D P E V K F N W Y V D G VE V H N A K T K P R E E Q Y N S T Y R V V S V L T V L H Q D W L N G K EY K C K V S N K A L P A P I E K T I S K A K G Q P R E P Q V Y T L P P SR D E L T K N Q V S L T C L V K G F Y P S D I A V E W E S N G Q P E N NY K T T P P V L D S D G S F F L Y S K L T V D K S R W Q Q G N V F S C SV M H E A L H N H Y T Q K S L S L S P G K G G G G G A Q L A D H G Q C IR W P W M C P P E G W E

Determination of pH Condition for Lyophilized Fc-Myo Binding Peptide

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC),which was stability-indicating at elevated temperatures. A pH screenstudy was designed and performed to determine the optimal formulation pHin the liquid state prior to the lyophilization. Protein was formulatedin pH 4.5, 4.75, 5.0, 5.5 and 6.0 with buffer agents of acetate andhistidine and sucrose as the stabilizer (or lyo protectant). Theformulated vials were stored at 29 C for up to 1 year. Stability wasmonitored using SE-HPLC. The aggregation rate constants were calculatedfor each of the formulation conditions. The aggregation rate at pH 4.5was found to be the minimum, therefore pH 4.5 was selected as thepreferred formulation pH condition.

Lyophilized Fc-Myo Binding Peptide Buffer Agent Study at 30 mg/mL.

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC),which was stability-indicating. A 30 mg/mL dosage form as investigatedusing three different buffering agent: 10 mM glutamate, 10 mM histidineand 10 mM succinate at pH 4.5. All formulations contain 0.004%polysorbate 20.

For the formulation development work, purified bulk material wasobtained in the 30 mg/mL frozen liquid formulation. The material wasdialyzed into the appropriate formulation buffers and lyophilized in aVirtis lyophilizer using a conservative cycle. Lyophilized proteinformulation displayed acceptable cake elegance. Upon reconstitutionformulations were clear Lyophilized Fc-Myo binding peptide was stored at4, 29, 37 and 52° C. The real time stability studies were carried out at4° C. and found to be comparable for these formulations for up to 3months. However at 52° C. storage 3 months, the histidine containingformulation was slightly better than the glutamate containingformulation. The succinate containing formulation was significantly lessstable than the other two formulations. Based on these results,histidine and glutamate were considered as the preferred buffer agentsfor the final Fc-Myo binding peptide formulation.

Lyophilized Fc-Myo Binding Peptide Excipient Study at 30 mg/ml:Stabilizing Effect of Sucrose, Trehalose and Hydroxyethyl Starch

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC),which was stability-indicating. To evaluate the effect of the presenceof trehalose, hydroxyethyl starch and sucrose on stability oflyophilized Fc-Myo binding peptide, Fc-Myo binding peptide wasformulated at 30 mg/mL in 10 mM glutamate buffer with 4% mannitol. Theconcentration of trehalose and sucrose used was 2.0%, 1% hydroxyethylstarch was added to the sucrose formulation to make a final formulationof 10 mM glutamate, 4% mannitol, 2% sucrose, 1% hydroxyethyl starch. Allformulations contain 0.004% polysorbate 20.

For the formulation development work, purified bulk material wasobtained in the 30 mg/mL frozen liquid formulation. The material wasdialyzed into the appropriate formulation buffers and lyophilized in aVirtis lyophilizer using a conservative cycle. Lyophilized proteinformulation displayed acceptable cake elegance. Upon reconstitutionformulations were clear.

The stability of these formulations was monitored using SE-HPLC.

Lyophilized Fc-Myo binding peptide was stored at 4, 29, 37 and 52° C.The real time shelf time condition stability (4° C.) was foundcomparable between these formulations for up to 3 months. However under52° C. storage condition for 3 months, the sucrose containingformulation was slightly better than the trehalose containingformulation. Addition of hydroxyethyl starch did not display anynegative impact on the stability. Based on these results, sucrose wasconsidered as the preferred stabilizer for the final Fc-Myo bindingpeptide formulation.

Lyophilized Fc-Myo binding Peptide Excipient Study at 30 mg/ml:Stabilizing Effect of Sucrose and Mannitol

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC),which was stability-indicating. To evaluate the effect of the presenceof variable amount of mannitol and sucrose on stability andreconstitution properties of lyophilized Fc-Myo binding peptide over themannitol range of 4.0 to 8% and the sucrose range of 1.0% to 4.0%,Fc-Myo binding peptide was formulated at 30 mg/mL in 10 mM glutamatebuffer. All formulations contain 0.004% polysorbate 20.

For the formulation development work, purified bulk material wasobtained in the 30 mg/mL frozen liquid formulation. The material wasdialyzed into the appropriate formulation buffers and lyophilized in aVirtis lyophilizer using a conservative cycle. Lyophilized proteinformulation displayed acceptable cake elegance. Upon reconstitutionformulations were clear.

The stability of these formulations was monitored using SE-HPLC method.Lyophilized Fc-Myo binding peptide was stored at 4, 29, 37 and 52° C.The real time shelf time condition stability (4° C.) was foundcomparable between these formulations for up to 3 months. However whenstored at 52° C. for 3 months, an increasing amount of sucrose was foundcontributing to the increase in stability against aggregation. Due to aconcern to maintain the isotonic condition for the final formulationwhich limits the total amount of disaccharides and to maintain a properratio of mannitol and sucrose to preserve the lyophilized cake property,4.0% mannitol and 2.0% sucrose were the preferred excipients for thefinal formulation.

Lyophilized Fc-Myo Binding Peptide Excipient Study at 1, 30, 85 mg/mL

The stability was assessed primarily by size-exclusion HPLC (SE-HPLC),which was stability-indicating. To evaluate the effect of the proteinconcentration on stability and reconstitution properties of lyophilizedFc-Myo binding peptide, Fc-Myo binding peptide was formulated at 1, 30,85 mg/mL in 10 mM glutamate buffer with 4% mannitol and 2% sucrose. Allformulations contain 0.004% polysorbate 20.

For the formulation development work, purified bulk material wasobtained in the 30 mg/mL frozen liquid formulation. The material wasbuffer exchanged into the appropriate formulation buffers using UF/DFand lyophilized in a Virtis lyophilizer using a conservative cycle.Lyophilized protein formulation displayed acceptable cake elegance. Uponreconstitution formulations were clear.

The stability of these formulations was monitored using SE-HPLC method.Lyophilized Fc-Myo binding peptide was stored at 4, 29, 37° C. The realtime shelf time condition stability (4° C.) was found comparable betweenthese formulations for up to 6 months. The stability is likelyacceptable for all the concentrations studied as the commercial productformulation.

CONCLUSION

4.0% mannitol and 2.0% sucrose have provided sufficient cake and proteinstability as confirmed by 12-month shelf study at 4° C. Therefore, 10 mMhistidine, 4.0% mannitol, 2.0% sucrose at pH 4.5 and 10 mM glutamate,4.0% mannitol, 2.0% sucrose at pH 4.5 can be used for formulating 1 to100 mg/mL Fc-Myo binding peptide.

The present invention has been described in terms of particularembodiments found or proposed to comprise preferred modes for thepractice of then invention. It will be appreciated by those of ordinaryskill in the art that, in light of the present disclosure, numerousmodifications and changes can be made in the particular embodimentsexemplified without departing from the intended scope of the invention.

1. A lyophilized therapeutic peptibody composition comprising a buffer,a bulking agent, a stabilizing agent, and optionally a surfactant;wherein said buffer is comprised of a pH buffering agent in a range ofabout 5 mM to about 20 mM and wherein the pH is in a range of about 3.0to about 8.0; wherein said bulking agent is at a concentration of about0% to about 4.5% w/v; wherein said stabilizing agent is at aconcentration of about 0.1% to about 20% w/v; wherein said surfactant isat a concentration of about 0.004% to about 0.4% w/v; and wherein saidtherapeutic peptibody comprises a structure set out in Formula I,[(X¹)_(a)—F¹—(X²)_(b)]-(L¹)_(c)-WSP_(d)  Formula I wherein: F¹ is an Fcdomain; X¹ is selected from P¹-(L²)_(e) P²-(L³)_(f)-P¹-(L²)_(e)—P³-(L⁴)_(g)-P²-(L³)_(f)-P¹-(L²)_(e)- andP⁴-(L⁵)_(h)-P³-(L⁴)_(g)-P²-(L³)-P¹-(L²)_(e)- X² is selected from:-(L²)_(e)-P¹, -(L²)_(e)-P¹-(L³)_(f)-P²,-(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³, and-(L²)_(g)-P¹-(L³)_(f)-P²-(L⁴)_(g)-P³-(L⁵)_(h)-P⁴ wherein P¹, P², P³, andP⁴ are each independently sequences of pharmacologically activepeptides; L¹, L², L³, L⁴, and L⁵ are each independently linkers; a, b,c, e, f, g, and h are each independently 0 or 1, provided that at leastone of a and b is 1; d is 0, 1, or greater than 1; and WSP is a watersoluble polymer, the attachment of which is effected at any reactivemoiety in F¹.
 2. The composition of claim 1 wherein said therapeuticpeptibody comprises a structure set out in Formula II[X¹—F¹]-(L¹)_(c)-WSP_(d)  Formula I wherein the Fc domain is attached atthe C-terminus of X¹, and zero, one or more WSP is attached to the Fcdomain, optionally through linker L¹.
 3. The composition of claim 1wherein said therapeutic peptibody comprises a structure set out inFormula III[F¹—X²]-(L¹)_(c)-WSP_(d)  Formula III wherein the Fc domain is attachedat the N-terminus of X², and zero, one or more WSP is attached to the Fcdomain, optionally through linker L¹.
 4. The composition of claim 1wherein said therapeutic peptibody comprises a structure set out inFormula IV[F¹-(L¹)_(e)-P¹]-(L¹)_(c)-WSP_(d)  Formula IV wherein the Fc domain isattached at the N-terminus of -(L¹)_(c)-P¹ and zero, one or more WSP isattached to the Fc domain, optionally through linker L¹.
 5. Thecomposition of claim 1 wherein said therapeutic peptibody comprises astructure set out in Formula V[F¹-(L¹)_(e)-P¹-(L²)_(f)-P²]-(L¹)_(c)-WSP_(d)  Formula V wherein the Fcdomain is attached at the N-terminus of -L¹-P¹-L²-P² and zero, one ormore WSP is attached to the Fc domain, optionally through linker L¹. 6.The composition of any one of claims 1 through 5 wherein saidtherapeutic peptibody is a multimer.
 7. The composition of claim 6wherein said therapeutic peptibody is a dimer.
 8. The composition of anyone of claims 1 through 5 and 7 wherein P¹, P², P³ and/or P⁴ areindependently selected from a peptide set out in any one of Tables 4through
 38. 9. The composition of claim 8 wherein P¹, P², P³ and/or P⁴have the same amino acid sequence.
 10. The composition of any one ofclaims 1 through 5 and 7 wherein the Fc domain is set out in SEQ IDNO:1.
 11. The composition of claim 8 wherein the Fc domain is set out inSEQ ID NO:1.
 12. The composition of any one of claims 1 through 5 and 7wherein WSP is PEG.
 13. The composition of claim 8 wherein WSP is PEG.14. The composition of any one of claims 1 through 5 and 7 wherein theFc domain is set out in SEQ ID NO:1 and WSP is PEG.
 15. The compositionof claim 8 wherein the Fc domain is set out in SEQ ID NO:1 and WSP isPEG.
 16. The composition of claim 14 wherein PEG has a molecular weightof between about 2 kDa and 100 kDa.
 17. The composition of claim 16wherein said PEG has a molecular weight of between about 6 kDa and 25kDa.
 18. The composition of claim 12, wherein said composition comprisesat least 50% PEGylated therapeutic peptibody.
 19. The composition ofclaim 18 comprising at least 75% PEGylated therapeutic peptibody. 20.The composition of claim 18 comprising at least 85% PEGylatedtherapeutic peptibody.
 21. The composition of claim 18 comprising atleast 90% PEGylated therapeutic peptibody.
 22. The composition of claim18 comprising at least 95% PEGylated therapeutic peptibody.
 23. Thecomposition of any one of claims 1 through 5 and 7 wherein said pHbuffering agent is selected from the group consisting of glycine,histidine, glutamate, succinate, phosphate, acetate, and aspartate. 24.The composition of claim 8 wherein said pH buffering agent is selectedfrom the group consisting of glycine, histidine, glutamate, succinate,phosphate, acetate, and aspartate.
 25. The composition of any one ofclaims 1 through 5 and 7 wherein said bulking agent selected from thegroup consisting of mannitol, glycine, sucrose, dextran,polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol,trehalose, or xylitol.
 26. The composition of claim 8 wherein saidbulking agent selected from the group consisting of mannitol, glycine,sucrose, dextran, polyvinylpyrolidone, carboxymethylcellulose, lactose,sorbitol, trehalose, or xylitol.
 27. The composition of any one ofclaims 1 through 5 and 7 wherein said stabilizing agent selected fromthe group consisting of sucrose, trehalose, mannose, maltose, lactose,glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose,glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL,poly-hydroxy compounds, including polysaccharides such as dextran,starch, hydroxyethyl starch, cyclodextrins, N-methyl pyrollidene,cellulose and hyaluronic acid, sodium chloride.
 28. The composition ofclaim 8 wherein said stabilizing agent selected from the groupconsisting of sucrose, trehalose, mannose, maltose, lactose, glucose,raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine,fructose, mannitol, sorbitol, glycine, arginine HCL, poly-hydroxycompounds, including polysaccharides such as dextran, starch,hydroxyethyl starch, cyclodextrins, N-methylpyrollidene, cellulose andhyaluronic acid, sodium chloride.
 29. The composition of any one ofclaims 1 through 5 and 7 wherein said surfactant selected from the groupconsisting of sodium lauryl sulfate, dioctyl sodium sulfosuccinate,dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosinesodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt,sodium cholate hydrate, sodium deoxycholate, glycodeoxycholic acidsodium salt, benzalkonium chloride or benzethonium chloride,cetylpyridinium chloride monohydrate, hexadecyltrimethylammoniumbromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, TritonX-114, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylenehydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate,polysorbate 20, 40, 60, 65 and 80, soy lecithin, DOPC, DMPG, DMPC, andDOPG; sucrose fatty acid ester, methyl cellulose and carboxymethylcellulose.
 30. The composition of claim 8 wherein said surfactantselected from the group consisting of sodium lauryl sulfate, dioctylsodium sulfosuccinate, dioctyl sodium sulfonate, chenodeoxycholic acid,N-lauroylsarcosine sodium salt, lithium dodecyl sulfate,1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodiumdeoxycholate, glycodeoxycholic acid sodium salt, benzalkonium chlorideor benzethonium chloride, cetylpyridinium chloride monohydrate,hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12,digitonin, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60,glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, soy lecithin,DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methyl celluloseand carboxymethyl cellulose.
 31. The composition of any one of claims 1through 5 and 7 wherein the therapeutic peptibody concentration isbetween about 0.25 mg/mL and 250 mg/mL.
 32. The composition of claim 8wherein the therapeutic peptibody concentration is between about 0.25mg/mL and 250 mg/mL.
 33. The composition of any one of claims 1 through5 and 7 wherein said pH buffering agent is 10 mM histidine and whereinthe pH is 5.0; wherein said bulking agent is 4% w/v mannitol; whereinsaid stabilizing agent is 2% w/v sucrose; and wherein said surfactant is0.004% w/v polysorbate-20.
 34. The composition of claim 33 wherein P¹comprises a sequence set forth in Table
 6. 35. The composition of claim34 wherein the therapeutic peptibody concentration is 0.5 mg/mL.
 36. Thecomposition of any one of claims 1 through 5 and 7 wherein said pHbuffering agent is 10 mM histidine and wherein the pH is 7.0; whereinsaid bulking agent is 4% w/v mannitol; wherein said stabilizing agent is2% w/v sucrose; and wherein said surfactant is 0.01% w/v polysorbate-20.37. The composition of claim 36 wherein P¹ comprises a sequence setforth in Table
 32. 38. The composition of claim 37 wherein thetherapeutic peptibody concentration is 30 mg/mL.
 39. The composition ofany one of claims 1 through 5 and 7 wherein said pH buffering agent is20 mM histidine and wherein the pH is 5.0; wherein said bulking agent is3.3% w/v mannitol; wherein said stabilizing agent is 2% w/v sucrose; andwherein said surfactant is 0.01% w/v polysorbate-20.
 40. The compositionof claim 39 wherein P¹ comprises a sequence set forth in Table
 4. 41.The composition of claim 40 wherein the therapeutic peptibodyconcentration is 100 mg/mL.
 42. The composition of any one of claims 1through 5 and 7 wherein said pH buffering agent is 10 mM histidine andwherein the pH is 5.0; wherein said bulking agent is 2.5% w/v mannitoland wherein said stabilizing agent is 3.5% w/v sucrose.
 43. Thecomposition of claim 42 wherein P¹ comprises a sequence set forth inTable
 31. 44. The composition of claim 43 wherein the therapeuticpeptibody concentration is 30 mg/mL.
 45. The composition of any ofclaims 1 through 5 and 7 wherein the composition is selected from thegroup consisting of: a) 10 mM histidine, pH 4.7, 4% mannitol and 2%sucrose, with and without 0.004% polysorbate-20; b) 10 mM histidine, pH5, 4% mannitol and 2% sucrose, with and without 0.004% polysorbate-20;c) 10 mM glutamate, pH 4.5, 4% mannitol and 2% sucrose with and without0.004% polysorbate-20; d) 10 mM succinate, pH 4.5, 4% mannitol and 2%sucrose, 0.004% polysorbate-20; e) 10 mM glutamate, pH 4.5, 9% sucrose,0.004% polysorbate-20; f) 10 mM glutamate, pH 4.5, 4% mannitol, 2%sucrose, 1% hydroxyethyl starch, 0.004% polysorbate-20; g) 5 mMglutamate, pH 4.5, 2% mannitol, 1% sucrose, 0.004% polysorbate-20; andh) 10 mM glutamate, pH 4.5, 4% mannitol, 2% trehalose, 0.004%polysorbate-20.
 46. The composition according to claim 45 wherein P¹comprises a sequence set forth in Tables 21-24.
 47. The composition ofclaim 46 wherein the therapeutic peptibody concentration is selectedfrom the group consisting of 1, 30, 85, and 100 mg/mL.
 48. A method formaking a lyophilized therapeutic peptibody comprising the steps of: a)preparing a solution of a buffer, a bulking agent, a stabilizing agent,and optionally a surfactant; wherein said buffer is comprised of a pHbuffering agent in a range of about 5 mM to about 20 mM and wherein thepH is in a range of about 3.0 to about 8.0; wherein said bulking agentis at a concentration of about 2.5% to about 4% w/v; wherein saidstabilizing agent is at a concentration of about 0.1% to about 5% w/v;wherein said surfactant is at a concentration of about 0.004% to about0.04% w/v; and b) lyophilizing said therapeutic peptibody; wherein saidtherapeutic peptibody comprises a structure set out in Formula I,[(X¹)_(a)—F¹—(X²)_(b)]-(L¹)_(c)-WSP_(d)  Formula I wherein: F¹ is an Fcdomain; X¹ is selected from P¹-(L²)_(e)— P²-(L³)_(f)-P¹-(L²)_(e)—P³-(L⁴)_(g)-P²-(L³)_(f)-P¹-(L²)_(e)- and P⁴(L⁵)_(h)-P³-(L⁴)_(g)-P²-(L³)_(f)-P¹-(L²)_(e)— X² is selected from:-(L²)_(e)-P¹, -(L²)_(e)-P¹-(L³)_(f)-P²,-(L²)—P¹-(L³)_(f)-P²-(L⁴)_(g)-P³, and -(L²)_(e)-P¹-(L³)_(f)-P²-(L⁴)_(g)P³-(L⁵)_(h)-P⁴ wherein P¹, P², P³, and P⁴ are each independentlysequences of pharmacologically active peptides; L¹, L¹, L³, L⁴, and Lare each independently linkers; a, b, c, e, f, g, and h are eachindependently 0 or 1, provided that at least one of a and b is 1; d is0, 1, or greater than 1; and WSP is a water soluble polymer, theattachment of which is effected at any reactive moiety in F¹.
 49. Themethod of claim 48 wherein said therapeutic peptibody comprises astructure set out in Formula II[X¹—F¹]-(L¹)_(c)-WSP_(d)  Formula II wherein the Fc domain is attachedat the C-terminus of X¹, and zero, one or more WSP is attached to the Fcdomain, optionally through linker L¹.
 50. The method of claim 48 whereinsaid therapeutic peptibody comprises a structure set out in Formula III[F¹—X²]-(L¹)_(c)-WSP_(d)  Formula III wherein the Fc domain is attachedat the N-terminus of X², and zero, one or more WSP is attached to the Fcdomain, optionally through linker L¹.
 51. The method of claim 48 whereinsaid therapeutic peptibody comprises a structure set out in Formula IV[F¹-(L¹)_(e)-P¹]-(L¹)_(c)-WSP_(d)  Formula IV wherein the Fc domain isattached at the N-terminus of -(L¹)_(c)-P¹ and zero, one or more WSP isattached to the Fc domain, optionally through linker L¹.
 52. The methodof claim 48 wherein said therapeutic peptibody comprises a structure setout in Formula V[F¹-(L¹)_(e)-P¹-(L²)_(f)-P²]-(L¹)_(c)-WSP_(d)  Formula V wherein the Fcdomain is attached at the N-terminus of -L¹-P¹-L²-P² and zero, one ormore WSP is attached to the Fc domain, optionally through linker L¹. 53.The method of any one of claims 48 through 52 wherein said therapeuticpeptibody is a multimer.
 54. The method of claim 53 wherein saidtherapeutic peptibody is a dimer.
 55. The method of any one of claims 48through 52 and 54 wherein P¹, P², P³ and/or P⁴ are independentlyselected from a peptide set out in any one of Tables 4 through
 38. 56.The method of claim 55 wherein P¹, P², P³ and/or P⁴ have the same aminoacid sequence.
 57. The method of any one of claims 48 through 52 and 54wherein the Fc domain is set out in SEQ ID NO:1.
 58. The method of claim55 wherein the Fc domain is set out in SEQ ID NO:1.
 59. The method ofany one of claims 48 through 52 and 54 wherein WSP is PEG.
 60. Themethod of claim 55 wherein WSP is PEG.
 61. The method of any one ofclaims 48 through 52 and 54 wherein the Fc domain is set out in SEQ IDNO:1 and WSP is PEG.
 62. The method of claim 55 wherein the Fc domain isset out in SEQ ID NO:1 and WSP is PEG.
 63. The method of claim 61wherein PEG has a molecular weight of between about 2 kDa and 100 kDa.64. The method of claim 63 wherein said PEG has a molecular weight ofbetween about 6 kDa and 25 kDa.
 65. The method of claim 64, wherein saidcomposition comprises at least 50% PEGylated therapeutic peptibody. 66.The method of claim 65 comprising at least 75% PEGylated therapeuticpeptibody.
 67. The method of claim 65 comprising at least 85% PEGylatedtherapeutic peptibody.
 68. The method of claim 65 comprising at least90% PEGylated therapeutic peptibody.
 69. The method of claim 65comprising at least 95% PEGylated therapeutic peptibody.
 70. The methodof any one of claims 48 through 52 and 54 wherein said pH bufferingagent is selected from the group consisting of glycine, histidine,glutamate, succinate, phosphate, acetate, and aspartate.
 71. The methodof claim 55 wherein said pH buffering agent is selected from the groupconsisting of glycine, histidine, glutamate, succinate, phosphate,acetate, and aspartate.
 72. The method of any one of claims 48 through52 and 54 wherein said bulking agent selected from the group consistingof mannitol, glycine, sucrose, dextran, polyvinylpyrolidone,carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. 73.The method of claim 55 wherein said bulking agent selected from thegroup consisting of mannitol, glycine, sucrose, dextran,polyvinylpyrolidone, carboxymethylcellulose, lactose, sorbitol,trehalose, or xylitol.
 74. The method of any one of claims 48 through 52and 54 wherein said stabilizing agent selected from the group consistingof sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose,cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose,mannitol, sorbitol, glycine, arginine HCL, poly-hydroxy compounds,including polysaccharides such as dextran, starch, hydroxyethyl starch,cyclodextrins, N-methyl pyrollidene, cellulose and hyaluronic acid,sodium chloride.
 75. The method of claim 55 wherein said stabilizingagent selected from the group consisting of sucrose, trehalose, mannose,maltose, lactose, glucose, raffinose, cellobiose, gentiobiose,isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol,glycine, arginine HCL, poly-hydroxy compounds, including polysaccharidessuch as dextran, starch, hydroxyethyl starch, cyclodextrins,N-methylpyrollidene, cellulose and hyaluronic acid, sodium chloride. 76.The method of any one of claims 48 through 52 and 54 wherein saidsurfactant selected from the group consisting of sodium lauryl sulfate,dioctyl sodium sulfosuccinate, dioctyl sodium sulfonate,chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecylsulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate,sodium deoxycholate, glycodeoxycholic acid sodium salt, benzalkoniumchloride or benzethonium chloride, cetylpyridinium chloride monohydrate,hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12,digitonin, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60,glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, soy lecithin,DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methyl celluloseand carboxymethyl cellulose.
 77. The method claim 55 wherein saidsurfactant selected from the group consisting of sodium lauryl sulfate,dioctyl sodium sulfosuccinate, dioctyl sodium sulfonate,chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecylsulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate,sodium deoxycholate, glycodeoxycholic acid sodium salt, benzalkoniumchloride or benzethonium chloride, cetylpyridinium chloride monohydrate,hexadecyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12,digitonin, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60,glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, soy lecithin,DOPC, DMPG, DMPC, and DOPG; sucrose fatty acid ester, methyl celluloseand carboxymethyl cellulose.
 78. The method of any one of claims 48through 52 and 54 wherein the therapeutic peptibody concentration isbetween about 0.25 mg/mL and 250 mg/mL.
 79. The method of claim 55wherein the therapeutic peptibody concentration is between about 0.25mg/mL and 250 mg/mL.
 80. The method of any one of claims 48 through 52and 54 wherein said pH buffering agent is 10 mM histidine and whereinthe pH is 5.0; wherein said bulking agent is 4% w/v mannitol; whereinsaid stabilizing agent is 2% w/v sucrose; and wherein said surfactant is0.004% w/v polysorbate-20.
 81. The method of claim 80 wherein P¹comprises a sequence set forth in Table
 6. 82. The method of claim 81wherein the therapeutic peptibody concentration is 0.5 mg/mL.
 83. Themethod of any one of claims 48 through 52 and 54 wherein said pHbuffering agent is 10 mM histidine and wherein the pH is 7.0; whereinsaid bulking agent is 4% w/v mannitol; wherein said stabilizing agent is2% w/v sucrose; and wherein said surfactant is 0.01% w/v polysorbate-20.84. The method of claim 83 wherein P¹ comprises a sequence set forth inTable
 32. 85. The method of claim 84 wherein the therapeutic peptibodyconcentration is 30 mg/mL.
 86. The method of any one of claims 48through 52 and 54 wherein said pH buffering agent is 20 mM histidine andwherein the pH is 5.0; wherein said bulking agent is 3.3% w/v mannitol;wherein said stabilizing agent is 2% w/v sucrose; and wherein saidsurfactant is 0.01% w/v polysorbate-20.
 87. The method of claim 86wherein P¹ comprises a sequence set forth in Table
 4. 88. The method ofclaim 87 wherein the therapeutic peptibody concentration is 100 mg/mL.89. The method of any one of claims 48 through 52 and 54 wherein said pHbuffering agent is 10 mM histidine and wherein the pH is 5.0; whereinsaid bulking agent is 2.5% w/v mannitol; and wherein said stabilizingagent is 3.5% w/v sucrose.
 90. The method of claim 89 wherein P¹comprises a sequence set forth in Table
 31. 91. The method of claim 90wherein the therapeutic peptibody concentration is 30 mg/mL.
 92. Themethod of any of claims 48 through 52 and 54 wherein the composition isselected from the group consisting of: a) 10 mM histidine, pH 4.7, 4%mannitol and 2% sucrose, with and without 0.004% polysorbate-20; b) 10mM histidine, pH 5, 4% mannitol and 2% sucrose, with and without 0.004%polysorbate-20; c) 10 mM glutamate, pH 4.5, 4% mannitol and 2% sucrosewith and without 0.004% polysorbate-20; d) 10 mM succinate, pH 4.5, 4%mannitol and 2% sucrose, 0.004% polysorbate-20; e) 10 mM glutamate, pH4.5, 9% sucrose, 0.004% polysorbate-20; f) 10 mM glutamate, pH 4.5, 4%mannitol, 2% sucrose, 1% hydroxyethyl starch, 0.004% polysorbate-20; g)5 mM glutamate, pH 4.5, 2% mannitol, 1% sucrose, 0.004% polysorbate-20;and h) 10 mM glutamate, pH 4.5, 4% mannitol, 2% trehalose, 0.004%polysorbate-20.
 93. The method according to claim 92 wherein P¹comprises a sequence set forth in Tables 21-24.
 94. The method of claim93 wherein the therapeutic peptibody concentration is selected from thegroup consisting of 1, 30, 85, and 100 mg/mL.
 95. The method of any oneof claims 48 through 52 and 54 further comprising, prior tolyophilization, the steps of: b) adjusting the pH of the solution to apH between about 4.0 and about 8.0; c) preparing a solution containingsaid therapeutic peptibody; d) buffer exchanging the solution of step(c) into the solution of step (b); e) adding an appropriate amount of asurfactant; and f) lyophilizing the mixture from step (e).
 96. A methodfor preparing a reconstituted therapeutic peptibody compositioncomprising the steps of: a) lyophilizing a therapeutic peptibodycomposition according to any one of claims 1 through 5 and 7; and b)reconstituting said lyophilized therapeutic peptibody composition.
 97. Amethod for preparing a reconstituted therapeutic peptibody compositioncomprising the steps of: a) lyophilizing a therapeutic peptibodycomposition according to claim 8; and b) reconstituting said lyophilizedtherapeutic peptibody composition.
 98. A kit for preparing an aqueouspharmaceutical composition comprising a first container having alyophilized therapeutic peptibody composition of any one of claims 1through 5 and 7, and a second container having a physiologicallyacceptable solvent for the lyophilized composition.
 99. A kit forpreparing an aqueous pharmaceutical composition comprising a firstcontainer having a lyophilized therapeutic peptibody composition ofclaim 8, and a second container having a physiologically acceptablesolvent for the lyophilized composition.